Abstract
The purple anoxygenic photosynthetic bacterium Rhodobacter capsulatus is capable of growing in aerobic or anaerobic conditions, in the dark or using light, etc. Achieving versatile metabolic adaptations from respiration to photosynthesis requires the use of tetrapyrroles such as heme and bacteriochlorophyll, in order to carry oxygen, to transfer electrons, and to harvest light energy. A third tetrapyrrole, cobalamin (vitamin B12), is synthesized and used as a cofactor for many enzymes. Heme, bacteriochlorophyll, and vitamin B12 constitute three major end products of the tetrapyrrole biosynthetic pathway in purple bacteria. Their respective synthesis involves a plethora of enzymes, several that have been characterized and several that are uncharacterized, as described in this review. To respond to changes in metabolic requirements, the pathway undergoes complex regulation to direct the flow of tetrapyrrole intermediates into a specific branch(s) at the expense of other branches of the pathway. Transcriptional regulation of the tetrapyrrole synthesizing enzymes by redox conditions and pathway intermediates is reviewed. In addition, we discuss the involvement of several transcription factors (RegA, CrtJ, FnrL, AerR, HbrL, Irr) as well as the role of riboswitches. Finally, the interdependence of the tetrapyrrole branches on each other synthesis is discussed.
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1 Introduction
Cyclic tetrapyrroles encompass porphyrins, such as heme, chlorophyll and bacteriochlorophyll (Bchl), and porphynoids, which are more reduced. The porphynoid class consists of the corrinoids (cobalamin), siroheme, heme d 1, and coenzyme F430 (Frankenberg et al. 2003). Many species are capable of synthesizing numerous tetrapyrrole end products. One of the best studied organisms is Rhodobacter capsulatus, an anoxygenic photosynthetic bacterium of the α-proteobacteria subfamily. It is capable of growing under a variety of different environmental conditions such as dark aerobic, dark and light anaerobic. This versatile metabolism requires the capability of synthesizing the tetrapyrroles heme and Bchl that are needed for respiratory or photosynthetic growth. Heme is needed for electron transfer during both respiration and photosynthesis while Bchl is the major pigment involved in collecting and converting light energy into biochemical energy during photosynthesis. In addition to synthesizing heme and Bchl, R. capsulatus also synthesizes cobalamin [vitamin B12 (vitB12)]. VitB12 and its derivatives are the most complex tetrapyrrole synthesized in nature and are involved in various cellular functions such as DNA repair and methionine synthesis (Martens et al. 2002). The synthesis of siroheme in R. capsulatus strain has been reported as well (Olmo-Mira et al. 2006).
The four tetrapyrrole end products synthesized by R. capsulatus are derived from different branches that split off from a common tetrapyrrole biosynthetic pathway “core” (Fig. 13.1). A common trunk of the pathway used by all three branches involves a multistep conversion of δ-aminolevulinic acid (δ-ALA) into uroporphyrinogen III (uro’gen III). At uro’gen III, the “corrin branch” splits off leading to the synthesis of cobalamin. Uro’gen III is also the potential substrate for siroheme synthesis. The central core of the pathway continues to synthesize intermediates up to protoporphyrin IX (proto IX) that are common to both the heme and Bchl branches. At proto IX, the heme and Bchl branches differentiate either through the insertion of Fe to form heme or the insertion of Mg to form Mg–proto IX which is the first committed step in the Mg branch that leads to the production of Bchl.
The biosynthesis of heme, vitB12, and Bchl is tightly controlled in order to achieve an adequate Bchl/heme/vitB12 ratio to meet the metabolic requirements of the cell. Heme and cobalamin levels appear to be maintained at rather stable levels while the amount of Bchl swings dramatically depending on the presence or absence of environmental oxygen. Moreover, a number of the intermediates in each of the three branches are potentially toxic as they can generate reactive singlet oxygen as a byproduct of light absorption. Tuning the relative amount of Bchl, heme, and vitB12 as a function of the metabolic requirements, while coping with the potential toxicity of intermediate products, requires this pathway to be highly regulated. Early work on the regulation of pigment synthesis in purple bacteria was initiated in the 1950s by Cohen-Bazire (Cohen-Bazire et al. 1957). More recently it has been demonstrated that the tetrapyrrole biosynthetic pathway in purple bacteria is highly complex and its regulation extremely sophisticated. This review is centered on the model species R. capsulatus and attempts to identify the actors involved in this pathway, both synthesizing enzymes and regulators.
2 Topology of Genes Involved in Synthesis of Tetrapyrroles
Examination of the complete genome of R. capsulatus has enabled the identification of the genes involved in each branch of tetrapyrrole synthesis (cf. annotations available at http://www.ergo-light.com and http://onco.img.cas.cz/rhodo/results/index.html). While genes involved in the vitB12 and Bchl synthesis are found in large operons, genes encoding the enzymes involved in heme synthesis are scattered over the genome (Young et al. 1989; McGoldrick et al. 2002; Warren et al. 2002; Smart et al. 2004). This feature is not unusual, though a few organisms show an operon organization for some of their hem genes (Avissar and Moberg 1995). The one exception in R. capsulatus is the hemC and hemE genes that are divergently transcribed and separated by 111 bp (Smart et al. 2004).
3 The Universal Trunk: From δ-ALA to Urog’en III
3.1 Feeding the Universal Trunk: The Synthesis of δ-ALA
The biosynthetic pathway to produce uro’gen III from δ-ALA is highly conserved in nature and is therefore considered universal. However, there are two different pathways to synthesize δ-ALA, which is the precursor of all known tetrapyrroles. One is the C-5 pathway where two enzymatic reactions transform glutamyl-tRNAGlu into δ-ALA. The other is the C-4 or Shemin pathway that involves the condensation of glycine and succinyl-CoA to produce δ-ALA, CO2, and CoA (Avissar and Moberg 1995; Frankenberg et al. 2003). As a typical representative of the α-proteobacteria subfamily, R. capsulatus synthesizes δ-ALA using the C-4 pathway. This reaction is performed by δ-ALA synthase (EC 2.3.1.37, ALAS), encoded by the hemA gene, previously identified and cloned in 1988 (Biel et al. 1988). R. capsulatus ALAS is a piridoxal-5′-phosphate-dependent homodimer, consisting of two 44 kDa subunits. It shows 49% identity and 70% similarity with the human erythroid-specific ALAS. The structure, solved in 2005 by Astner et al., provided insight on mutations that generate dysfunctional forms of the human eALAS (Astner et al. 2005). Moreover, despite a very low primary sequence identity of 18%, ALAS shows remarkable three-dimensional homology with the glutamate-1-semialdehyde-2,1-aminomutase (GSAM) of Thermosynechococcus elongatus. The GSAM is the last enzyme of the C-5 pathway and, as such, synthesizes δ-ALA. It is likely that the C-4 pathway originated from the evolution of GSAM into ALAS, followed by the loss of the C-5 pathway in common ancestors of α-proteobacteria and of early mitochondria (Schulze et al. 2006). ORF encoding a putative GSAM, hemL, was found in the genome of R. capsulatus. In prokaryotes, the occurrence of two uncoupled δ-ALA biosynthetic pathways was shown in Streptomyces nodosus subsp. asukaensis, where the C-5 pathway produces δ-ALA for heme synthesis, while the C-4 pathway generates δ-ALA targeted to secondary metabolism (Petricek et al. 2006). In R. capsulatus there is no evidence for an alternative δ-ALA synthesis pathway as the first enzyme of the C-5 pathway, glutamyl-tRNA reductase, is not found in the genome. Furthermore, the annotation of this ORF as a hemL gene has to be confirmed experimentally as GSAM-encoding genes share a great deal of similarity with genes coding for other types of aminotranferases (Panek and O’Brian 2002). Interestingly, when a δ-ALA-requiring mutant containing a single insertion was characterized, it was found that a low level (10%) of the ALAS activity of the parental strain remained (Wright et al. 1987). Finally, the synthesis of δ-ALA appears as one of the crucial regulation points of the tetrapyrrole biosynthetic pathway, the transcription of hemA being up-regulated under low oxygen tension (Wright et al. 1991; Smart et al. 2004) and down-regulated in the presence of excess of exogenous heme (Smart and Bauer 2006).
3.2 Entering the Universal Trunk: Synthesis of Porphobilinogen
The first common step in tetrapyrrole synthesis is achieved by porphobilinogen synthase (EC 4.2.1.24, PBGS), or δ-ALA dehydratase, that converts two δ-ALA molecules into a monopyrrole porphobilinogen. In R. capsulatus, hemB encodes a 35.8 kDa protein which presents unusual characteristics among the PBGS studied so far. Indeed, most PBGS are known to be homooctameric metalloenzymes presenting a high variability regarding their cations requirement in terms of catalysis (Zn2+-, Mg2+-, or K+- binding site at the active site) and activity regulation, most of them containing an extra allosteric Mg2+-binding site. The biochemical characterization of R. capsulatus revealed that its enzyme is a homohexamer clearly independent of cations (Zn2+, Mg2+, K+) or chelators (EDTA, 1,10-phenathroline). R. capsulatus PBGS seems to switch between a highly active homohexameric form and a less active homodimeric form (Nandi and Shemin 1973; Bollivar et al. 2004). Regarding metal dependence, this feature was partially predicted from the primary sequence. Indeed, among 130 PBGS sequences representing the entire tree of life, only the ones from the Rhodobacter genus, namely R. capsulatus and R. sphaeroides, lack the cysteine-rich motif involved in Zn binding at the active site and the conserved arginine and glutamate residues that coordinate the allosteric Mg. It is postulated that PBGS containing Zn at the active site evolved to other configurations (metal free, Mg- and/or K-containing active site) during the set up of the photosynthetic process and the early synthesis of Bchl. Indeed, Zn is a competitor of Mg in the metallation step of proto IX (Jaffe 2003; Frère et al. 2005). If the PBGSs of the Rhodobacter genus fit well in this model regarding their active site, the evolutionary pressure(s) that allowed them to totally lose metal content, including the Mg in the allosteric site, remain(s) unknown. Moreover, despite porphobilinogen accumulation under low oxygen tension in R. capsulatus, the activity of PBGS is unregulated by oxygen, proto IX, and hemin in R. capsulatus. In addition, no or only a slight increase in hemB transcription was observed upon decreasing the oxygen tension (Biel et al. 2002; Smart et al. 2004). However, the conversion of δ-ALA into porphobilinogen seems to be a major control point in the tetrapyrrole biosynthesis pathway (Biel 1992). This may be achieved by controlling the level of porphobilinogen through the availability of the substrate of PBGS, i.e., δ-ALA. Indeed, the vast majority of the δ-ALA synthesized is transformed into aminohydroxyvalerate by an oxygen-dependent δ-ALA dehydrogenase (Biel et al. 2002). Lastly, although not occurring in a manner as dramatic as for hemA, hemC, or hemE, it was shown that the transcription of hemB is repressed in the presence of exogenous heme (Smart and Bauer 2006).
3.3 Reaching the First Crossroad: Synthesis of the Tetrapyrrole Ring, Uro’gen III
The transformation of monopyrrole porphobilinogen into the cyclic tetrapyrrole uro’gen III is catalyzed by the enzymes porphobilinogen deaminase (EC 4.2.1.24, PBGD) and uro’gen III synthase (4.2.1.75, UROS) encoded by hemC and hemD, respectively. During the first step, PBGD polymerizes four molecules of porphobilinogen into a linear tetrapyrrole, pre-uro’gen or 1-hydroxymethylbilane, and four NH3. The main product, pre-uro’gen, is very unstable and, if exposed to solvent, will be converted chemically into a toxic cyclic porphyrin, uro’gen I. To avoid toxicity pre-uro’gen is passed to UROS that presumably interacts with PBGD to limit exposure of the cell to free pre-uro’gen (Shoolingin-Jordan 1995). UROS catalyzes the formation of uro’gen III, by circularizing the linear tetrapyrrole while inverting ring D. An asymmetric ring D is a signature of biologically functional porphyrins.
PBGD was purified from R. capsulatus and the N-terminal sequence was determined in order to identify the corresponding gene, hemC. The latter was also expressed heterologously in Escherichia coli, confirming that this ORF is sufficient to produce an active form of PBGD (Biel et al. 2002). No biochemical characterization of the R. capsulatus enzyme has been performed so far, but analysis of the sequence indicates that hemC corresponds to a 34.1 kDa protein with 47% homology with PBGDs from E. coli and Pseudomonas aeruginosa and 43% with the Bacillus subtilis homolog. In addition, previous characterizations of PBGDs highlighted the use of a unique cofactor, dipyrromethane which consists of two porphobilinogen molecules. This cofactor acts as a primer during the polymerization of a hexapyrrole, which finally releases the tetrapyrrolic reaction product and the intact cofactor. R. capsulatus PBGD contains the conserved cysteine residue that is involved in cofactor binding. More work has been done regarding the genetics. Indeed, characterization of a hemC mutant confirmed that it was incapable of synthesizing either siroheme or vitB12 and grew only in media supplemented with cysteine and methionine, in addition to hemin (Biel et al. 2002). As well, hemC expression was shown to be regulated by oxygen, with a fivefold increase of the transcription level when changing from aerobic to anaerobic growth conditions (Smart et al. 2004). Finally, the transcription of hemC was repressed two-thirds upon the addition of exogenous hemin (Smart and Bauer 2006).
UROS is only very poorly characterized unclear as it has never been purified from R. capsulatus. A high variability in the primary sequences of UROSs has made it difficult to identify the hemD gene in the R. capsulatus genome. In addition, the lack of genetic organization such as a hemCD operon or a hem gene cluster, as in E. coli or B. subtilis, renders the task even harder (Avissar and Moberg 1995). Recently, an ORF in the R. capsulatus genome was annotated as encoding a UROS but because hemD genes have been miss-annotated in many other organisms, experimental work has to be undertaken to confirm this annotation (Panek and O’Brian 2002).
4 The Porphyrin Branch: From Uro’gen III to Heme
4.1 The Synthesis of Coproporphyrinogen III and Its Puzzling Secretion
The first step after the uro’gen III crossroad consists in the synthesis of coproporphyrinogen III (copro’gen III). This activity is performed by uro’gen III decarboxylase (EC 4.1.1.37, UROD) which catalyzes the sequential decarboxylation of the four acetate side chains into methyl groups. The reaction is ordered, starting with the ring D and then decarboxylating rings A, B, and finally ring C. Numerous UROD-encoding genes from various biological sources have been identified showing a rather high level of conservation and presumably a common mechanism of action (Frankenberg et al. 2003; Heinemann et al. 2008). The R. capsulatus gene encoding UROD, hemE, was identified in 1995 (Ineichen and Biel 1995). The deduced protein sequence shows more than 34% identity with previously characterized UROD and contains a signature sequence of this class of enzyme (PXWXMRQAGR) at the amino-terminal. No biochemical work has been performed with R. capsulatus UROD but a bit is known regarding the genetics and its regulation. An interesting feature of hemE is its linkage to hemC, while the other hem genes are scattered on the genome. These two genes are divergently transcribed and separated by an intergenic region of 111 bp. Furthermore, the transcription of hemE is strongly dependent on redox conditions, as seen using a hemE::lacZ fusion. Expression of hemE increases by 112- and 52-fold under semi-aerobic and anaerobic conditions, respectively (Smart et al. 2004). As with hemC, the transcription level of hemE is decreased by two-thirds in the presence of exogenous heme (Smart and Bauer 2006).
An interesting phenomenon about this step of the pathway is that the cells excrete copious amounts of the product of the reaction, copro’gen III under certain growth conditions. Cooper (1956, 1963) highlighted this feature in R. capsulatus, using cells grown under iron starvation or with excess exogenous methionine. More recently, various R. capsulatus mutants, either unable to synthesize Mg–protoporphyrin monomethyl ester or lacking c-type cytochromes, were studied and showed a similar phenotype. Moreover, it was discovered that coproporphyrin is excreted as a complex with a 66 kDa protein (Biel and Biel 1990; Biel 1991) that turned out to be the major outer membrane porin (Bollivar and Bauer 1992).
4.2 Reaching the Second Crossroad: The Synthesis of Protoporphyrin IX
The antepenultimate and penultimate steps of heme synthesis are achieved by copro’gen III oxidase (EC 1.3.99.22, CPO) and proto’gen IX oxidase (EC 1.3.3.4, PPO), respectively. These steps lead to the formation of protoporphyrin IX which is a precursor for the synthesis of heme and Bchl. Oxygen-dependent and oxygen-independent CPOs conventionally named HemF and HemN (or HemZ), respectively, are known to catalyze this reaction. HemF is prevalent in eukaryotes and in a few bacterial groups (primarily cyanobacteria and proteobacteria) while HemN is widely distributed among prokaryotes. These two forms of CPOs share no obvious primary sequence homology indicating that they arose from distinct evolutionary processes. Both enzymes convert copro’gen III into proto’gen IX by decarboxylating the propionate side chains of rings A and B consecutively to yield vinyl groups (Panek and O’Brian 2002; Frankenberg et al. 2003; Heinemann et al. 2008). Careful examination of the genome of R. capsulatus revealed no HemF-encoding gene but three ORFs that may code for putative HemNs. This is in contrast to the genome of the phylogenetically related species R. sphaeroides that contains one hemF and three hemN genes (locus tags in img.jgi.doe.gov: RSP_0682, RSP_0699, RSP_1224, RSP_0317).
HemN contains a [4Fe–4S] cluster for catalysis and requires S-adenosyl-l-methionine (SAM) as a cofactor or co-substrate. As such, this enzyme belongs to a family of “radical SAM” enzymes that carry the signature sequence CXXXCXXC, where the cysteine residues are involved in the coordination of the Fe atoms (Layer et al. 2005). This motif is present in the deduced protein sequences of the three putative CPOs. Occurrence of multiple hemN genes in one organism has been reported and several bacterial oxygen-independent CPOs have been shown to be expressed and active in the presence of oxygen (Rompf et al. 1998; Schobert and Jahn 2002). The expression of hemN1, designated hemZ in the cited articles, was studied as a function of aeration, which showed only a moderate influence. Indeed, a roughly twofold variation was recorded, with a maximum transcription level under semi-aerobic conditions (Smart et al. 2004). In addition, the presence of exogenous heme under these growth conditions decreased the expression of the gene by twofold factor, bringing it back to its basal level of transcription (Smart and Bauer 2006). Finally, in silico analysis of α-proteobacterial genomes revealed an “iron-rhodo-box” in the promoter region of the hemN2 gene. Thus, hemN2 is predicted to be part of the iron regulon (Rodionov et al. 2006).
The next step in the pathway is the aromatization of proto’gen IX by removal of six electrons to yield proto IX. This is performed by proto’gen IX oxidase (PPO) that exists as two forms, oxygen-dependent (HemY) and oxygen-independent (HemG). Most of our knowledge about PPOs comes from studies of eukaryotic enzymes that are oxygen dependent and inhibited by diphenyl ether herbicides in plants (Dailey 2002; Heinemann et al. 2008). In prokaryotes, the situation is very unclear as the PPO-encoding gene is unidentifiable in many genomes and, so far, hemG seems to be limited to six genera within the γ-proteobacteria group (Panek and O’Brian 2002; Frankenberg et al. 2003). As is the case with many other heme-synthesizing prokaryotes, a PPO-encoding gene has not been identified in the genome of either R. capsulatus or R. sphaeroides (cf. img.jgi.doe.gov). Even if the oxidation of proto’gen IX into proto IX can occur chemically, an enzyme-mediated catalysis is most probably required. Indeed, on the one hand, hemY and hemG mutants have been shown to be heme deficient in B. subtilis and E. coli, respectively (Panek and O’Brian 2002). On the other hand, this oxidation has to occur under anaerobic conditions as well, when the need for Bchl peaks. So there must be either an unknown PPO-encoding gene or an already known gene carrying out such an activity. The ERGO annotation of the R. capsulatus genome recently indicated an ORF encoding a “NAD (FAD) utilizing dehydrogenase with a possible PPO activity.” This has yet to be experimentally confirmed.
4.3 Delivering the Final Product: The Synthesis of Heme
The synthesis of heme from proto IX is performed by the enzyme ferrochelatase (EC 4.99.1.1, FC) that chelates ferrous iron and inserts it into the center of the tetrapyrrole ring. The core of the enzyme is highly conserved between bacteria, plants, and mammals, although bacterial FCs do not contain [2Fe–2S] clusters (Frankenberg et al. 2003; Heinemann et al. 2008). Interestingly, FC and the anaerobic cobalt chelatase CbiK are both class 2 metal ion chelatases that exhibit a similar three-dimensional structure (Schubert et al. 2002). Recently, Masoumi et al. confirmed in vivo what Koch et al. predicted in silico that PPO and FC form a stable PPO–FC complex (Koch et al. 2004; Masoumi et al. 2008). Such a configuration may ensure the channeling of the potentially toxic proto IX to FC. Finally, it has been suggested that this complex might include the CPO to form a tripartite association CPO–PPO–FC (Dailey 2002; Koch et al. 2004).
In R. capsulatus, the FC-encoding gene, hemH, was described and cloned in the 1990s with the deduced protein sequence exhibiting 41% (59%) and 21% (44%) identity (similarity) with E. coli and B. subtilis FCs (Kanazireva and Biel 1996). Moreover, homologous expression of a second copy of hemH causes a dramatic reduction of the tetrapyrrole pool, with 2.3-fold and 5-fold decreases of the porphyrin and the Bchl concentrations, respectively. In addition, the ALAS activity is also decreased 2.3-fold under these conditions (Kanazireva and Biel 1995). This phenotype is a signature of negative feedback by heme operating on the entire pathway, as has been studied more recently (Smart and Bauer 2006). The transcription of hemH was analyzed as well, highlighting a unique responsive behavior to oxygen among the other R. capsulatus hem genes. Indeed, while the expression level is rather constant between aerobic and anaerobic conditions, it is threefold lower under semi-aerobic conditions (Smart et al. 2004). Finally, in semi-aerobic conditions, hemH transcription was found to be independent of the exogenously added heme (Smart and Bauer 2006).
4.4 Regulation of hem Gene Expression
4.4.1 RegA
The RegA–RegB system is a global regulator in R. capsulatus that has been shown to regulate synthesis of numerous cellular processes as a function of redox conditions (reviewed in Elsen et al. 2004; Wu and Bauer 2008). As shown in Fig. 13.2, studies of the transcription of various hem genes in a regA mutant strain highlighted that hem genes are part of the RegA–RegB regulon (Smart et al. 2004). Indeed, the most significant effect was observed on hemE and hemH where transcription was activated 5- to 10-fold by RegA. A strong activating role of RegA was also observed with hemA but only in semi-aerobic and anaerobic conditions. Moderate influences were reported on hemC and hemZ. Finally, the transcription of hemB does not seem to be regulated by RegA (Smart et al. 2004). Overall, the dramatic transcriptional activation of hemE and hemH highlights the crucial intervention of the RegA–RegB system at key steps of the tetrapyrrole synthetic pathway. Activating the transcription of these genes ensures a constant stock of mRNA for a quick synthesis of UROD and FC when the flow of tetrapyrrole synthesis needs to be directed toward the porphyrin branch rather than the corrin branch and when heme needs to be produced at the expense of Bchl. Finally, the RegA-induced increase in the transcription of hemA under semi-aerobic and anaerobic conditions underlines a function of the RegA–RegB system, namely stimulation of the production of the general tetrapyrrole precursor δ-ALA in coordination with the synthesis of the pigment-binding proteins of the photosystem.
4.4.2 CrtJ
Present in the photosynthesis gene cluster is a redox-responding transcription factor called CrtJ or PpsR depending upon the species. Studies have demonstrated that CrtJ/PpsR is an aerobic repressor of heme (hem), Bchl (bch), and carotenoid (crt) biosynthesis genes in R. capsulatus (Ponnampalam and Bauer 1997; Elsen et al. 1998; Smart et al. 2004). CrtJ also aerobically represses synthesis of light-harvesting II peptides (puc) that bind Bchl and carotenoids in these species (Ponnampalam et al. 1995). CrtJ cooperatively binds to two copies of the palindromic sequence TGT-N12-ACA that is present in many hem promoter regions (Ponnampalam and Bauer 1997; Elsen et al. 1998; Ponnampalam et al. 1998; Smart et al. 2004).
The characterization of a crtJ mutant strain has revealed its involvement in the transcriptional control of several hem genes, summarized in Fig. 13.2 (Smart et al. 2004). The strongest regulation is the activation of hemH expression, whatever the redox condition is. CrtJ is also required to induce the translation of hemZ under semi-aerobic conditions. In the same redox conditions, CrtJ exhibits a dual role of repressor of hemC and activator of hemE at the same time. This is puzzling as these genes share a common promoter region, containing a CrtJ-binding site (Smart et al. 2004). Elucidating how, in constant redox conditions, the interaction with this promoter induces two opposite events will be a challenge.
4.4.3 FnrL
The redox regulator FnrL appeared to have only moderate effect on transcription of hem genes (Fig. 13.2), as either an activator or a repressor, depending on the gene and/or on redox conditions (Smart et al. 2004). This makes it hard to clearly define its mode of action and function in the regulation of tetrapyrrole synthesis. On the one hand, FnrL significantly represses hemA in aerobic conditions and hemB and hemC in semi-aerobic and anaerobic conditions. On the other hand, it dramatically activates the transcription of hemE in semi-aerobic conditions. FnrL seems to be mostly involved in the early steps of the tetrapyrrole synthesis, the universal trunk, and the first step of the porphyrin branch. It was shown in other photosynthetic bacteria, such as R. sphaeroides and Rubrivivax gelatinosus, to have a crucial role in the transcription of hemZ, which did not appear to occur in R. capsulatus (Smart et al. 2004; Ouchane et al. 2007).
4.4.4 AerR
The aerobic repressor AerR (Dong et al. 2002) has a limited although important role in the transcriptional regulation of the tetrapyrrole synthesis, consisting in the activation of hemE and hemH whatever the redox conditions and the repression of hemC in semi-aerobic conditions are (Fig. 13.2). Overall, AerR seems to back up CrtJ in its target genes, with the exception of hemZ (Smart et al. 2004).
4.4.5 HbrL
The heme-binding regulatory LysR-type (HbrL) protein was identified in 2006 from the screening of a cosmid library for suppressors defective in heme synthesis using hemB as a reporter (Smart and Bauer 2006). From these experiments, an 804 bp ORF was isolated and cloned and the deduced amino acid sequence showed a LysR-type transcriptional regulator type (LTTR) signature: A helix-turn-helix DNA-binding domain at the N-terminal followed by an LTTR substrate-binding motif or coinducer-binding motif. In addition to being ubiquitous in prokaryotes, the LTTRs represent the largest family of DNA-binding protein. They regulate functions as diverse as metabolism, cell division, quorum sensing, oxidative stress, virulence, nitrogen fixation, etc. (Maddocks and Oyston 2008). As shown in Fig. 13.2, HbrL appears to be involved in the heme-mediated control of the expression of hemA, hemB, and hemN1 (hemZ). Whereas it acts as a strong activator of hemA and hemN1 in the absence of exogenous heme, it is a moderate repressor of hemB. Recombinant HbrL was obtained by heterologous expression in E. coli and purification. It exhibited heme-binding properties either in vivo by supplementing the expression medium with δ-ALA or in vitro by adding heme to the crude lysate containing the apoprotein. Finally, recombinant HbrL was found to interact with the promoter region of the genes it regulates and the DNA-binding properties were affected by the presence or absence of heme (Smart and Bauer 2006). Interestingly, out of seven strains from the laboratory freezer, only four displayed an intact HbrL-encoding gene (unpublished observation).
4.4.6 Irr
The iron response regulator (Irr) is a transcription factor related to the ferric-uptake regulator (Fur) family of metalloregulators. It has been extensively studied in Bradyrhizobium japonicum where it is a global regulator of iron homeostasis that acts in particular as a repressor of heme synthesis in iron-limited conditions. It is constitutively expressed and its activity mostly controlled by post-translational modification. Indeed, its degradation is triggered by binding to both redox states of heme: The ferric form binds to an N-terminal heme regulatory motif (HRM), while the ferrous form interacts with a histidine-rich domain (Small et al. 2009). In the genome of R. capsulatus, an ORF was annotated as a Fur transcription factor (rcc02670 in http://onco.img.cas.cz/rhodo and RRC01140 in http://www.ergo-light.com/). Analysis of the sequence revealed that it belongs to the Irr subgroup rather than to the Fur one stricto sensu, as seen from divergence in key residues involved in metal coordination in the Fur group (Rudolph et al. 2006). Compared to the well-characterized B. japonicum Irr, it does not carry any HRM, which indicates a presumably different mechanism of action. On the other hand, the DNA-binding helix is very well conserved. It is noteworthy that, among the α-proteobacteria subfamily, R. capsulatus is the only representative that carries Irr as the sole regulator involved in the iron-responsive network. The usual master regulators Fur and RirA are indeed missing (Rodionov et al. 2006). This fact indicates a potential major role of Irr in R. capsulatus regarding the management of iron metabolism. Preliminary studies have shown that an irr-deleted strain of R. capsulatus presents abnormal levels of heme, approximately 25% less and 25% more than the wild-type strain in aerobic and photosynthetic conditions, respectively (unpublished data).
5 The Porphynoid Branch
5.1 The Siroheme Sub-branch: The Other Fe Branch
Siroheme, the prosthetic group of various reductases, was reported to occur in R. capsulatus strain E1F1. A 17 kb region contains genes encoding an assimilatory nitrate reduction system, which consists of (i) putative regulatory genes nsrR and nasTS; (ii) an ABC-type nitrate transporter coded by nasFED; (iii) genes coding for the apo-nitrate and apo-nitrite reductases, nasA and nasB; (iv) a gene encoding a siroheme synthase, cysG, responsible for synthesis of the nitrite reductase cofactor (Pino et al. 2006). Although the siroheme synthase was not biochemically studied, the nitrite reductase NasB has been heterologously expressed in E. coli and purified recombinant protein exhibited a siroheme spectral signature (Olmo-Mira et al. 2006). CysG (EC 2.1.1.107) was characterized in Salmonella enterica and in E. coli where it was shown to be a bimodular homodimer that catalyzes the four reactions that converts uro’gen III into siroheme (Stroupe et al. 2003).
5.2 The Corrin Sub-branch: From Uro’gen III to VitB12
5.2.1 An Unusual Pathway
Cobalamin synthesis requires more than 30 enzymatic steps with genes that encode these enzymes taking up approximately 1% of a typical bacterial genome (Roth et al. 1993; Martens et al. 2002). There are two distinct cobalamin biosynthesis pathways that exist in different microorganisms: the well-studied aerobic pathway, represented by Pseudomonas denitrificans, and the partially resolved anaerobic pathway, represented by Salmonella typhimurium, Bacillus megaterium, and Propionibacterium shermanii (Scott and Roessner 2002). The two pathways diverge at precorrin-2, a compound synthesized by dimethylation of uro’gen III (Raux et al. 1999). These two pathways primarily differ in their requirement for molecular oxygen and the timing of cobalt insertion. In the aerobic pathway, molecular oxygen is incorporated before the ring contraction, where the carbon bridge (C20) between the rings A and D is excised, and cobalt chelation occurs nine steps after the synthesis of precorrin-2. In contrast, in the anaerobic pathway, cobalt is inserted directly into precorrin-2. Ring contraction is likely facilitated by the different oxidation states of the cobalt ion (Martens et al. 2002; Warren et al. 2002). The existence of distinct cobalamin synthesis pathways is also reflected at the genetic level with organisms containing genes unique to one pathway or the other. Interestingly, R. capsulatus carries genetic hallmarks for an aerobic pathway although cobalamin synthesis occurs under both aerobic and anaerobic conditions (McGoldrick et al. 2002). A cobG gene, encoding a mono-oxygenase, is missing in R. capsulatus. This mono-oxygenase catalyzes the contraction of the corrin ring, which is a characteristic reaction in the aerobic pathway. Instead, an isofunctional enzyme, CobZ, was identified (McGoldrick et al. 2005), which may mediate the ring contraction process under both aerobic and anaerobic conditions. Details of enzymes at additional steps of this pathway are beyond the scope of this review but can be obtained from a recent review of this complex branch in the pathway by Warren (Warren and Deery 2009).
5.2.2 cob Gene Regulation
5.2.2.1 Riboswitch: Regulation at the Post-transcriptional Level
The regulation of cobalamin biosynthesis is mostly reported to occur post-transcriptionally via an RNA structure called a riboswitch. Riboswitches are metabolite-binding regions located within the 5′-UTR of messenger RNAs that function as regulatory elements for target genes (Mandal et al. 2003). Riboswitches are widespread in prokaryotes and interact with various target metabolites. In B. subtilis, seven different metabolites, including guanine, adenine, lysine, thiamine pyrophosphate, FMN, SAM, and adenosylcobalamin, regulate at least 68 genes via a riboswitch mechanism (Mandal et al. 2003). In cobalamin biosynthesis, adenosylcobalamin is known to be an effector regulating translation of several cobalamin genes (Nahvi et al. 2002; Rodionov et al. 2003). A B12-binding element exists within the 5′-UTR of btuB mRNA in E. coli and within the 5′-UTR of cobalamin biosynthesis operon in S. typhimurium that binds adenosylcobalamin (Nahvi et al. 2004). At elevated adenosylcobalamin concentrations, the translation of cobalamin transport protein (BtuB) and cob operon is repressed by adenosylcobalamin binding to the B12 box (Nahvi et al. 2004). Such interactions induce a conformational change in the mRNA secondary structure at the B12 box that prevents ribosome binding (Nou and Kadner 2000). Rodionov et al. (2003) employed the combination of a comparative approach of gene regulation, positional clustering, and phylogenetic profiling for identifying cobalamin-regulated biosynthesis/transport genes. They identified a conserved RNA structure called the B12 element that is widely distributed in eubacteria. For example, 13 B12 elements are present in UTR regions of the R. capsulatus genome with potential target genes, including cobW, cbiMNQO, btuFCD, btuD, and btuB, as well as upstream of several non-cobalamin biosynthesis/transport genes (Rodionov et al. 2003).
5.2.2.2 Transcriptional Regulation
Examples of cobalamin genes regulated at the transcriptional level are not abundant. In S. typhimurium, the global regulators Crp/cAMP and ArcA/ArcB are responsible for indirect redox and carbon controls of cobalamin biosynthesis by controlling synthesis of the positive regulatory protein, PocR. PocR subsequently uses propanediol as an inducer of cob gene expression (Rondon and Escalante-Semerena 1992, 1996). Other global regulators directly regulating cob genes are not known at present.
Recently, we observed that the global RegA–RegB signal transduction cascade not only controls expression of the Bchl and heme branches but also controls expression of enzymes at two steps in the cobalamin branch, the cobK gene and the cobWNHIJ operon (Li 2009). Thus, expression of cobalamin enzymes appears to be regulated by feedback control via a riboswitch mechanism as well as in response to redox via the RegA–RegB signal transduction cascade.
6 The Bchl Branch: From Proto IX to Bchl
6.1 The Bchl Biosynthesis Gene Cluster
Analysis of genes involved in the Mg branch of the tetrapyrrole biosynthesis pathway was primarily advanced by the work of Marrs and coworkers who used a generalized transducing agent (GTA) from R. capsulatus to map the location of Bchl biosynthesis genes (Marrs 1974; Yen and Marrs 1976). These studies established that carotenoid and Bchl biosynthesis genes were present in the R. capsulatus chromosome in a tight linkage order. The transduction mapping studies were followed by the isolation of an R′ plasmid that was shown by marker rescue and transposition mapping analyses to contain all of the essential genetic information needed to synthesize Bchl (Marrs 1981; Biel and Marrs 1983; Taylor et al. 1983; Zsebo and Hearst 1984). Structural and functional information regarding these loci was refined by complete sequence analysis of the R′ by Hearst and coworkers (Alberti et al. 1995) and by construction of defined interposon mutations in each of the sequenced open reading frames (Giuliano et al. 1988; Young et al. 1989; Yang and Bauer 1990; Bollivar et al. 1994a, b). Similar clustering of Bchl biosynthesis genes occurs in other purple bacterial species (Igarashi et al. 2001; Jaubert et al. 2004).
Several of the enzymes encoded by one or more of the Bchl biosynthesis genes have been expressed in E. coli and shown to exhibit enzymatic activity at specific steps of the Mg branch. The first committed step of the Mg branch involves insertion of Mg into proto IX by the enzyme Mg chelatase (EC 6.6.1.1) to form Mg–proto IX. Gibson et al. (1995) were the first to successfully heterologously express subunits of Mg chelatase encoded by the bchH, bchI, and bchD genes in E. coli to definitively establish the subunit composition of this enzyme. Activity is dependent on Mg, ATP, and proto IX. The multimeric Mg chelatase is a typical class 1 metal chelatase (Schubert et al. 2002) where BchH binds proto IX (Gibson et al. 1995) while the structures of BchI and BchD show that they both have ATPase domains of the AA+ class (Fodje et al. 2001).
The next step of the pathway involves SAM-dependent methylation of the carboxyl group of Mg–proto IX by the enzyme SAM Mg–proto IX-O-methyltransferase (EC 2.1.1.11) to form Mg–proto IX monomethyl ester (MPE). Confirmation that the R. capsulatus bchM encodes this enzyme occurred when this gene was heterologously expressed in E. coli with cell lysate extracts exhibiting activity (Bollivar et al. 1994a, b).
Oxidative cyclization to form the fifth ring of Bchl is catalyzed by the enzyme MPE oxidative cyclase (EC 1.14.13.81) that is coded by the bchE gene to form the product protochlorophyllide (PChlide). This enzyme has not been well characterized biochemically owing to the difficulty in observing activity in vitro. However, analysis of the R. capsulatus BchE peptide sequence indicates that it may be a SAM enzyme that also uses vitB12 as a cofactor (Gough et al. 2000). Indeed depletion of vitB12 in vivo leads to accumulation of MPE in this branch of the pathway (Gough et al. 2000).
Reduction of the D ring of PChlide to form chlorophyllide (Chlide) occurs by the enzyme NADPH–PChlide oxidoreductase (EC 1.3.1.33). In nature there are two unrelated enzymes that can catalyze this reduction. One is a light-dependent version that is present in cyanobacteria, alga, gymnosperms, and angiosperms, while the other is light-independent and present in anoxygenic bacteria, cyanobacteria, alga, and gymnosperms (Fujita 2003 and Bauer 2003; Heyes and Hunter 2005). The dark operative form is present in R. capsulatus, encoded by bchL, bchN, and bchB. Interestingly, it has a high degree of primary sequence similarity to nitrogenase (Fujita 2003 and Bauer 2003). Biochemical characterization of the R. capsulatus enzyme indicates that it contains iron sulfur centers and requires ATP, ferredoxin, and a reducing agent for catalysis (Fujita 2003 and Bauer 2003; Nomata et al. 2005). Reduction of ring B of Chlide by the enzyme Chlide a reductase (EC 1.3.1.75), encoded by bchX, bchY, and bchZ, also uses an enzyme that is very similar to dark PChlide reductase (Nomata et al. 2006).
There are several latter steps of the pathway that have not been biochemically characterized. The one exception is the esterification of the propionate on ring IV by the enzyme Bchl a synthase, encoded by bchG. This enzyme is membrane bound and difficult to isolate. However, it was heterologously expressed in E. coli with membrane fractions shown to exhibit this activity (Oster et al. 1997).
6.2 Regulation of Bchl Biosynthesis Gene Expression
6.2.1 CrtJ/PpsR
Studies have demonstrated that CrtJ/PpsR is an aerobic repressor of Bchl genes, bch, in R. capsulatus (Ponnampalam and Bauer 1997; Elsen et al. 1998). As discussed below, anaerobic induction of Bchl, carotenoid, and light-harvesting genes also requires phosphorylated RegA. So together, CrtJ and RegA regulate synthesis of Bchl biosynthesis genes by coordinating aerobic repression and anaerobic activation, respectively (Fig. 13.2).
CrtJ cooperatively binds to two copies of the palindromic sequence TGT-N12-ACA that is present in all of the characterized bch promoters (Alberti et al. 1995; Ponnampalam and Bauer 1997; Elsen et al. 1998; Ponnampalam et al. 1998). The palindromic sequence is found either 8 bp apart, or at sites that are distantly separated. For example, the R. capsulatus bchC promoter region has a CrtJ recognition palindrome that spans the –35 promoter region and a second CrtJ palindrome located 8 bp away that spans the –10 promoter region (Ponnampalam et al. 1998). Binding to these two palindromes is cooperative so if the 8 bp space between the two palindromes in the bchC promoter region is altered by the addition or deletion of just a few base pairs, then CrtJ is unable to bind to either palindrome effectively (Ponnampalam et al. 1998).
CrtJ also cooperatively binds to two palindromes at other promoters but these palindromes are separated by more than 100–150 bp (Elsen et al. 1998). An example of this type of binding occurs in the intergenic region between crtA and crtI that contains two promoters, one that is responsible for driving expression of the crtA–bchI–bchD operon and a second divergent promoter >100 bp away that is responsible for expression of the crtI–crtB operon (Elsen et al. 1998). The promoter for the crtA–bchI–-bchD transcript has a single CrtJ-binding site that spans the –10 promoter sequences while the crtI–crtB promoter also has a single CrtJ recognition sequence that spans the –35 recognition sequence. Cooperative binding of CrtJ to these two palindromes coordinately represses expression of both the crtA–bchI–bchD and crtI–crtB operons (Elsen et al. 1998). This affects synthesis of both Bchl and carotenoids since BchI and BchD are subunits of Mg chelatase (Bollivar et al. 1994a, b) and crtI and crtB code for phytoene dehydrogenase and phytoene synthase that are enzymes for the first two committed steps of carotenoid biosynthesis, respectively (Armstrong et al. 1990). Presumably, binding to distant sites involves looping of the DNA so that tetrameric CrtJ can bind cooperatively to both of the recognition palindromes (Elsen et al. 1998).
6.2.2 RegA–RegB
The Mg branch of the tetrapyrrole biosynthetic pathway is also anaerobically activated by the global two-component system RegA–RegB (Willett et al. 2007). Specifically, expression of the large bchEJG-orf428-bchP-idi operon that encodes numerous enzymes in Bchl biosynthesis, as well as an enzyme involved in carotenoid biosynthesis, requires phosphorylated RegA for maximal expression (Bollivar et al. 1994a, b; Alberti et al. 1995; Hahn et al. 1996; Suzuki et al. 1997; Bollivar 2006; Willett et al. 2007). In addition, phosphorylated RegA is also required for expression of the crtA–bchIDO operon that codes for early enzymes involved in the Bchl branch as well as early enzymes in the carotenoid biosynthesis pathway (Alberti et al. 1995).
7 Co-dependent Syntheses of Tetrapyrrole End Products
The trifurcated tetrapyrrole biosynthetic pathways not only share common early intermediates but also form an intricate network where the end products cobalamin and heme are involved in each others and Bchl biosyntheses. For example, in the Bchl branch, Gough et al. (2000) identified a cyclase encoded by bchE that catalyzes the conversion of MPE to PChlide. The sequence similarity between this MPE-cyclase and a cobalamin-dependent P-methylase from Streptomyces hygroscopicus indicates an involvement of cobalamin in MPE-cyclase activity. Although no in vitro cyclase assays have been reported, MPE-cyclase activity was demonstrated to be cobalamin dependent in vivo as cobalamin depletion or a bchE knockout results in an accumulation of MPE.
In addition to the dependence on cobalamin for Bchl biosynthesis, the synthesis of cobalamin is dependent on the presence of heme. Specifically, one of the most complicated steps in the R. capsulatus cobalamin branch is the corrin ring contraction that is catalyzed by the cofactor-rich enzyme, CobZ. CobZ contains heme as a cofactor as well as flavin and two Fe–S centers (McGoldrick et al. 2005). The dependence on heme as a cofactor of CobZ thus makes cobalamin synthesis heme dependent.
Finally, synthesis of heme is also cobalamin dependent as heme synthesis requires S-adenosylmethionine as a methyl group donor and its synthesis involves a cobalamin-dependent enzyme (Drennan et al. 1994; Layer et al. 2003; McGoldrick et al. 2005). The interdependent relationship among all three tetrapyrrole compounds as discussed in McGoldrick et al. (2005) suggests that codependence of these different tetrapyrrole branches on different tetrapyrrole end products is a form of regulation used to coordinate the levels of these different tetrapyrrole derivatives.
8 Conclusion
The tetrapyrrole biosynthetic pathway of R. capsulatus is quite complex involving several branches that are responsible for synthesis of heme, vitB12, and Bchl a. The flow of tetrapyrroles into each branch in the trifurcated pathway is highly regulated with an intricate network of transcriptional and post-transcriptional events controlling the synthesis of these various end products. This includes the control of enzyme activity by interaction with tetrapyrrole end products and transcriptional control either in response to redox poise or in response to an interaction with a tetrapyrrole. There is also a riboswitch control of translation via interaction of mRNA with a tetrapyrrole. Many players in this complex regulatory web have been identified, although it will not be surprising to find that there are additional players involved in controlling this complex pathway.
Among many challenges going forward will be to determine how multiple transcription factors coordinately regulate the synthesis of individual enzymes in the pathway. For example, the hemC promoter appears to be controlled by at least four different activators/repressors. Where these factors bind to the hemC promoter and how binding of individual transcription factors affects binding of other transcription factors remain to be determined. The study of this complex pathway has been an ongoing for over five decades and, owing to its complexity, will likely remain an area of focus for some time.
References
Alberti M, Burke DH, Hearst JE (1995) Anoxygenic photosynthetic bacteria. Kluwer Academic Publishers, Dordrecht
Armstrong GA, Schmidt A, Sandmann G et al. (1990) Genetic and biochemical characterization of carotenoid biosynthesis mutants of Rhodobacter capsulatus. J Biol Chem 265:8329–8338
Astner I, Schulze JO, van den Heuvel J et al (2005) Crystal structure of 5-aminolevulinate synthase, the first enzyme of heme biosynthesis, and its link to XLSA in humans. EMBO J 24:3166–3177
Avissar YJ, Moberg PA (1995) The common origins of the pigments of life – early steps of chlorophyll biosynthesis. Photosynth Res 44:221–242
Biel AJ, Marrs BL (1983) Transcriptional regulation of several genes for bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata in response to oxygen. J Bacteriol 156:686–694
Biel AJ (1991) Characterization of a coproporphyrin-protein complex from Rhodobacter capsulatus. FEMS Microbiol Lett 65:43–47
Biel AJ (1992) Oxygen-regulated steps in the Rhodobacter capsulatus tetrapyrrole biosynthetic pathway. J Bacteriol 174:5272–5274
Biel AJ, Canada K, Huang D et al (2002) Oxygen-mediated regulation of porphobilinogen formation in Rhodobacter capsulatus. J Bacteriol 184:1685–1692
Biel SW, Wright MS, Biel AJ (1988) Cloning of the Rhodobacter capsulatus hemA gene. J Bacteriol 170:4382–4384
Biel SW, Biel AJ (1990) Isolation of a Rhodobacter capsulatus mutant that lacks c-type cytochromes and excretes porphyrins. J Bacteriol 172:1321–1326
Bollivar DW, Bauer CE (1992) Association of tetrapyrrole intermediates in the bacteriochlorophyll a biosynthetic pathway with the major outer-membrane porin protein of Rhodobacter capsulatus. Biochem J 282:471–476
Bollivar DW, Jiang ZY, Bauer CE et al (1994a) Heterologous expression of the bchM gene product from Rhodobacter capsulatus and demonstration that it encodes S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase. J Bacteriol 176:5290–5296
Bollivar DW, Suzuki JY, Beatty JT et al (1994b) Directed mutational analysis of bacteriochlorophyll a biosynthesis in Rhodobacter capsulatus. J Mol Biol 237:622–640
Bollivar DW, Clauson C, Lighthall R et al (2004) Rhodobacter capsulatus porphobilinogen synthase, a high activity metal ion independent hexamer. BMC Biochem. doi: 10. 1186/1471-2091-5-17
Bollivar DW (2006) Recent advances in chlorophyll biosynthesis. Photosynth Res 90:173–194
Cohen-Bazire G, Sistrom WR, Stanier RY (1957) Kinetic studies of pigment synthesis by non-sulfur purple bacteria. J Cell Physiol 49:25–68
Cooper R (1956) The production of coproporphyrin precursors by a Rhodopseudomonas sp. Biochem J 63:25
Cooper R (1963) The biosynthesis of coproporphyrinogen, magnesium protoporphyrin monomethyl ester and bacteriochlorophyll by Rhodopseudomonas capsulata. Biochem J 89:100–108
Dailey HA (2002) Terminal steps of haem biosynthesis. Biochem Soc Trans 30:590–595
Dong C, Elsen S, Swem LR et al (2002) AerR, a second aerobic repressor of photosynthesis gene expression in Rhodobacter capsulatus. J Bacteriol 184:2805–2814
Drennan CL, Huang S, Drummond JT et al (1994) How a protein binds B12: A 3.0 A X-ray structure of B12-binding domains of methionine synthase. Science 266:1669–1674
Elsen S, Ponnampalam SN, Bauer CE (1998) CrtJ bound to distant binding sites interacts cooperatively to aerobically repress photopigment biosynthesis and light harvesting II gene expression in Rhodobacter capsulatus. J Biol Chem 273:30762–30769
Elsen S, Swem LR, Swem DL et al (2004) RegB/RegA, a highly conserved redox-responding global two-component regulatory system. Microbiol Mol Biol Rev 68:263–279
Fodje MN, Hansson A, Hansson M et al (2001) Interplay between an AAA module and an integrin I domain may regulate the function of magnesium chelatase. J Mol Biol 311:111–122
Frankenberg N, Moser J, Jahn D (2003) Bacterial heme biosynthesis and its biotechnological application. Appl Microbiol Biotechnol 63:115–127
Frère F, Reents H, Schubert WD et al (2005) Tracking the evolution of porphobilinogen synthase metal dependence in vitro. J Mol Biol 345:1059–1070
Fujita Y, Bauer CE (2003) The light-independent protochlorophyllide reductase: a nitrogenase-like enzyme catalyzing a key reaction for greening in the dark. In: Smith K, Kadish K, Guilard R (eds) The porphyrin handbook. Academic, Amsterdam, the Netherlands
Gibson LC, Willows RD, Kannangara CG et al (1995) Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli. Proc Natl Acad Sci USA 92:1941–1944
Giuliano G, Pollock D, Stapp H et al (1988) A genetic-physical map of the Rhodobacter capsulatus carotenoid biosynthesis gene cluster. Mol Gen Genet MGG 213:78–83
Gough SP, Petersen BO, Duus JO (2000) Anaerobic chlorophyll isocyclic ring formation in Rhodobacter capsulatus requires a cobalamin cofactor. Proc Natl Acad Sci USA 97:6908–6913
Hahn FM, Baker JA, Poulter CD (1996) Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase. J Bacteriol 178:619–624
Heinemann IU, Jahn M, Jahn D (2008) The biochemistry of heme biosynthesis. Arch Biochem Biophys 474:238–251
Heyes DJ, Hunter CN (2005) Making light work of enzyme catalysis: protochlorophyllide oxidoreductase. Trends Biochem Sci 30:642–649
Igarashi N, Harada J, Nagashima S et al (2001) Horizontal transfer of the photosynthesis gene cluster and operon rearrangement in purple bacteria. J Mol Evol 52:333–341
Ineichen G, Biel AJ (1995) Nucleotide sequence of the Rhodobacter capsulatus hemE gene. Plant Physiol 108:423
Jaffe EK (2003) An unusual phylogenetic variation in the metal ion binding sites of porphobilinogen synthase. Chem Biol 10:25–34
Jaubert M, Zappa S, Fardoux J et al (2004) Light and redox control of photosynthesis gene expression in Bradyrhizobium: dual roles of two PpsR. J Biol Chem 279:44407–44416
Kanazireva E, Biel AJ (1995) Cloning and overexpression of the Rhodobacter capsulatus hemH gene. J Bacteriol 177:6693–6694
Kanazireva E, Biel AJ (1996) Nucleotide sequence of the Rhodobacter capsulatus hemH gene. Gene 170:149–150
Koch M, Breithaupt C, Kiefersauer R et al (2004) Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis. EMBO J 23:1720–1728
Layer G, Moser J, Heinz DW et al (2003) Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes. EMBO J 22:6214–6224
Layer G, Kervio E, Morlock G et al (2005) Structural and functional comparison of HemN to other radical SAM enzymes. Biol Chem 386:971–980
Li K (2009) Regulation of and by cobalamin in Rhodobacter capsulatus. PhD thesis, Indiana University, BLoomington, Indiana
Maddocks SE, Oyston PCF (2008) Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology 154:3609–3623
Mandal M, Boese B, Barrick JE et al (2003) Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria. Cell 113:577–586
Marrs B (1974) Genetic recombination in Rhodopseudomonas capsulata. Proc Natl Acad Sci USA 71:971–973
Marrs B (1981) Mobilization of the genes for photosynthesis from Rhodopseudomonas capsulata by a promiscuous plasmid. J Bacteriol 146:1003–1012
Martens JH, Barg H, Warren MJ et al (2002) Microbial production of vitamin B12. Appl Microbiol Biotechnol 58:275–285
Masoumi A, Heinemann IU, Rohde M et al (2008) Complex formation between protoporphyrinogen IX oxidase and ferrochelatase during haem biosynthesis in Thermosynechococcus elongatus. Microbiology 154:3707–3714
McGoldrick H, Deery E, Warren M et al (2002) Cobalamin (vitamin B(12)) biosynthesis in Rhodobacter capsulatus. Biochem Soc Trans 30:646–648
McGoldrick HM, Roessner CA, Raux E et al (2005) Identification and characterization of a novel vitamin B12 (cobalamin) biosynthetic enzyme (CobZ) from Rhodobacter capsulatus, containing flavin, heme, and Fe-S cofactors. J Biol Chem 280:1086–1094
Nahvi A, Sudarsan N, Ebert MS et al (2002) Genetic control by a metabolite binding mRNA. Chem Biol 9:1043–1049
Nahvi A, Barrick JE, Breaker RR (2004) Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes. Nucleic Acids Res 32:143–150
Nandi DL, Shemin D (1973) δ-Aminolevulinic acid dehydratase of Rhodopseudomonas capsulata. Arch Biochem Biophys 158:305–311
Nomata J, Swem LR, Bauer CE et al (2005) Overexpression and characterization of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus. Biochim Biophys Acta 1708:229–237
Nomata J, Mizoguchi T, Tamiaki H et al (2006) A second nitrogenase-like enzyme for bacteriochlorophyll biosynthesis: reconstitution of chlorophyllide a reductase with purified X-protein (BchX) and YZ-protein (BchY-BchZ) from Rhodobacter capsulatus. J Biol Chem 281:15021–15028
Nou X, Kadner RJ (2000) Adenosylcobalamin inhibits ribosome binding to btuB RNA. Proc Natl Acad Sci USA 97:7190–7195
Olmo-Mira MF, Cabello P, Pino C et al (2006) Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1. Arch Microbiol 186:339–344
Oster U, Bauer CE, Rudiger W (1997) Characterization of chlorophyll a and bacteriochlorophyll a synthases by heterologous expression in Escherichia coli. J Biol Chem 272:9671–9676
Ouchane S, Picaud M, Therizols P et al (2007) Global regulation of photosynthesis and respiration by FnrL: the first two targets in the tetrapyrrole pathway. J Biol Chem 282:7690–7699
Panek H, O’Brian MR (2002) A whole genome view of prokaryotic haem biosynthesis. Microbiology 148:2273–2282
Petricek M, Petrickova K, Havlicek L et al (2006) Occurrence of two 5-aminolevulinate biosynthetic pathways in Streptomyces nodosus subsp. asukaensis is linked with the production of asukamycin. J Bacteriol 188:5113–5123
Pino C, Olmo-Mira F, Cabello P et al (2006) The assimilatory nitrate reduction system of the phototrophic bacterium Rhodobacter capsulatus E1F1. Biochem Soc Trans 34:127–129
Ponnampalam SN, Buggy JJ, Bauer CE (1995) Characterization of an aerobic repressor that coordinately regulates bacteriochlorophyll, carotenoid, and light harvesting-II expression in Rhodobacter capsulatus. J Bacteriol 177:2990–2997
Ponnampalam SN, Bauer CE (1997) DNA binding characteristics of CrtJ. A redox-responding repressor of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression in Rhodobacter capsulatus. J Biol Chem 272:18391–18396
Ponnampalam SN, Elsen S, Bauer CE (1998) Aerobic repression of the Rhodobacter capsulatus bchC promoter involves cooperative interactions between CrtJ bound to neighboring palindromes. J Biol Chem 273:30757–30761
Raux E, Schubert HL, Roper JM et al (1999) Vitamin B12: insights into biosynthesis’s mount improbable. Bioorg Chem 27:100–118
Rodionov DA, Vitreschak AG, Mironov AA et al (2003) Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes. J Biol Chem 278:41148–41159
Rodionov DA, Gelfand MS, Todd JD et al (2006) Computational reconstruction of iron- and manganese-responsive transcriptional networks in alpha-proteobacteria. PLoS Comput Biol. doi: 10.1371/journal.pcbi.0020163
Rompf A, Hungerer C, Hoffmann T et al (1998) Regulation of Pseudomonas aeruginosa hemF and hemN by the dual action of the redox response regulators Anr and Dnr. Mol Microbiol 29:985–997
Rondon MR, Escalante-Semerena JC (1992) The poc locus is required for 1,2-propanediol-dependent transcription of the cobalamin biosynthetic (cob) and propanediol utilization (pdu) genes of Salmonella typhimurium. J Bacteriol 174:2267–2272
Rondon MR, Escalante-Semerena JC (1996) In vitro analysis of the interactions between the PocR regulatory protein and the promoter region of the cobalamin biosynthetic (cob) operon of Salmonella typhimurium LT2. J Bacteriol 178:2196–2203
Roth JR, Lawrence JG, Rubenfield M et al (1993) Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium. J Bacteriol 175:3303–3316
Rudolph G, Hennecke H, Fischer HM (2006) Beyond the Fur paradigm: iron-controlled gene expression in rhizobia. FEMS Microbiol Rev 30:631–648
Schobert M, Jahn D (2002) Regulation of heme biosynthesis in non-phototrophic bacteria. J Mol Microbiol Biotechnol 4:287–294
Schubert HL, Raux E, Matthews MA et al (2002) Structural diversity in metal ion chelation and the structure of uroporphyrinogen III synthase. Biochem Soc Trans 30:595–600
Schulze JO, Schubert WD, Moser J et al (2006) Evolutionary relationship between initial enzymes of tetrapyrrole biosynthesis. J Mol Biol 358:1212–1220
Scott AI, Roessner CA (2002) Biosynthesis of cobalamin (vitamin B(12)). Biochem Soc Trans 30:613–620
Shoolingin-Jordan PM (1995) Porphobilinogen deaminase and uroporphyrinogen III synthase: structure, molecular biology, and mechanism. J Bioenerg Biomembr 27:181–195
Small SK, Puri S, O’Brian MR (2009) Heme-dependent metalloregulation by the iron response regulator (Irr) protein in Rhizobium and other Alpha-proteobacteria. Biometals 22:89–97
Smart JL, Willett JW, Bauer CE (2004) Regulation of hem gene expression in Rhodobacter capsulatus by redox and photosystem regulators RegA, CrtJ, FnrL, and AerR. J Mol Biol 342:1171–1186
Smart JL, Bauer CE (2006) Tetrapyrrole biosynthesis in Rhodobacter capsulatus is transcriptionally regulated by the heme-binding regulatory protein, HbrL. J Bacteriol 188:1567–1576
Stroupe ME, Leech HK, Daniels DS et al (2003) CysG structure reveals tetrapyrrole-binding features and novel regulation of siroheme biosynthesis. Nat Struct Biol 10:1064–1073
Suzuki JY, Bollivar DW, Bauer CE (1997) Genetic analysis of chlorophyll biosynthesis. Annu Rev Genet 31:61–89
Taylor DP, Cohen SN, Clark WG et al (1983) Alignment of genetic and restriction maps of the photosynthesis region of the Rhodopseudomonas capsulata chromosome by a conjugation-mediated marker rescue technique. J Bacteriol 154:580–590
Warren MJ, Raux E, Schubert HL et al (2002) The biosynthesis of adenosylcobalamin (vitamin B12). Nat Prod Rep 19:390–412
Warren MJ, Deery E. 2009. Vitamin B12 (cobalamin) biosynthesis in the purple bacteria. In: Hunter CN, Daldal F, Thurnauer MC, Beatty JT (eds) Advances in Photosynthesis and Respiration, vol 28. Springer, Dordrecht
Willett J, Smart JL, Bauer CE (2007) RegA control of bacteriochlorophyll and carotenoid synthesis in Rhodobacter capsulatus. J Bacteriol 189:7765–7773
Wright MS, Cardin RD, Biel AJ (1987) Isolation and characterization of an aminolevulinate-requiring Rhodobacter capsulatus mutant. J Bacteriol 169:961–966
Wright MS, Eckert JJ, Biel SW et al (1991) Use of a lacZ fusion to study transcriptional regulation of the Rhodobacter capsulatus hemA gene. FEMS Microbiol Lett 62:339–342
Wu J, Bauer CE (2008) RegB/RegA, A global redox-responding two-component regulatory system. In: Utsumi R (ed) Advances in experimental medicine and biology, vol 631. Landes Bioscience Eurekah, Georgetown
Yang ZM, Bauer CE (1990) Rhodobacter capsulatus genes involved in early steps of the bacteriochlorophyll biosynthetic pathway. J Bacteriol 172:5001–5010
Yen HC, Marrs B (1976) Map of genes for carotenoid and bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata. J Bacteriol 126:619–629
Young DA, Bauer CE, Williams JC et al (1989) Genetic evidence for superoperonal organization of genes for photosynthetic pigments and pigment-binding proteins in Rhodobacter capsulatus. Mol Gen Genet 218:1–12
Zsebo KM, Hearst JE (1984) Genetic-physical mapping of a photosynthetic gene cluster from R. capsulata. Cell 37:937–947
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Zappa, S., Li, K., Bauer, C.E. (2010). The Tetrapyrrole Biosynthetic Pathway and Its Regulation in Rhodobacter capsulatus . In: Hallenbeck, P. (eds) Recent Advances in Phototrophic Prokaryotes. Advances in Experimental Medicine and Biology, vol 675. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-1528-3_13
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