Introduction

Cytochrome P450(CYPs) enzymes are found ubiquitously in many living organisms, including humans, animals, plants, and bacteria. They play a crucial role in the metabolism of a wide variety of compounds, including drugs, toxins, and endogenous substrates such as steroids and fatty acids [1]. CYPs are heme-containing enzymes that use a heme iron center to catalyze reactions. The heme iron is coordinated to a cysteine residue in the enzyme, forming the heme-thiolate (Fe-S-Cys) complex. The heme-thiolate complex is responsible for the ability of CYPs to bind and activate molecular oxygen (O2) and to insert an oxygen atom into the substrate molecule. The majority of P450 enzymes employ NAD(P)H as a coenzyme, which undergoes dehydrogenation to form NAD(P)+ and release electrons. The electrons are then transferred to FMN, converting it into FMNH2 before being passed on to Fe-S protein and ultimately the heme center’s Fe atom. The oxygen atoms activated by Fe atom subsequently react with substrates [2].

CYPs are classified based on the electron transfer process involved in their catalytic cycle. There are five major classes of CYP enzymes (Fig. 1). Class I is a three-component system containing P450 enzymes, iron redox protein reductase (FDR) with a molecule of flavin adenine dinucleotide (FAD), and iron redox protein (FDX) with iron-sulfur clusters. The majority of P450 enzymes in this class are derived from prokaryotes and eukaryotic mitochondria [3]. Class II is a two-component system where the redox chaperone (i.e., P450 reductase CPR) carries both FAD and FMN binding sites. These enzymes are commonly derived from eukaryotes, including animals, plants, and fungi [4]. Class III is a one-component system, the most representative enzyme being P450BM3 from Bacillus megaterium (CYP102A). The P450BM3 domain fuses with the FAD/FMN binding sites in redox chaperones [5]. Class IV is also a one-component system that naturally fuses the P450 structural domain with the Fe2S2/FMN binding sites in the redox chaperone. Finally, Class V includes small naturally occurring P450 enzymes that do not require redox chaperones and obtain electrons directly from NADPH, such as nitric oxide reductase (P450nor) [6].

Fig. 1
figure 1

Classification of P450 enzymes. According to the relationship between P450 enzymes and chaperone proteins, P450 enzymes can be roughly divided into five classes. Class I is a three-component system, Class II is a two-component system, and Class III, Class IV, and Class V are one-component systems

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P450 enzymes display diverse catalytic activities, with C-H bond hydroxylation being the most significant. This process enables the oxidation of specific sites such as fatty acids, carbon ring compounds, and heterocyclic compounds for use in drug synthesis or other important compound production [7, 8]. For example, Dahlbäck et al. demonstrated that CYP105A2, in Streptomyces lividans, catalyzes selective hydroxylation of vitamin D3 at 25-position, and 25-OHVD3 is an important drug used to treat osteoporosis and chronic renal failure [9]. Yan et al. improved the conversion rate and regioselectivity of P450 enzyme-catalyzed C–H oxidation through protein engineering, signifying an important direction for P450 enzyme research [10].

CYP153A family (CYP153A) [11]are the important cytochrome P450 enzymes found in alkane-degrading bacteria. It has a broad substrate specificity and can oxidize compounds with diverse chemical structures [12] (Fig. 2). It is involved in the biosynthesis of a wide range of active compounds, including polycyclic aromatic hydrocarbons (PAHs), fatty acids, steroids, and drugs [13]. Additionally, it plays a crucial role in the initial step of the degradation pathway for these compounds within microorganisms by converting them into hydroxyl compounds that are subsequently metabolized by other enzymes. It is a low-cost and high-efficiency bioremediation reagent, which has broad application prospects in polluted environments [14].

Fig. 2
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Classification, structural analysis, molecular modification, and application prospects of CYP153A are discussed in the current review

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Overall, CYP153A is an important enzyme with diverse biotechnological applications, and its properties are the subject of ongoing research. In this review, we have summarized recent research about CPY153A family classification, function, structure, molecular modifications, and application in biocatalysis and synthetic biology.

Classification and function of CYP153A

CYP153A is a class of P450 enzymes family derived mainly from microorganisms with n-alkanes as their only carbon source [15, 16]. CYP153A has a heme-dependent monooxygenase core (CYP) with two additional redox proteins and/or domains. Furthermore, it has an iron-sulfur electron donor (FDX) and a reductase containing FDR. CYP153A can transfer electrons from NADPH to the monooxygenase active site [11, 12]and catalyze hydroxylation of medium n-alkanes, such as n-octane and n-hexane [17]. At present, many CYP153 family enzymes have been reported (Table 1).

Table 1 CYP153A classification and basal characteristics
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Using quantitative polymerase chain reaction (qPCR), Yong et al. found an upregulation of the CYP153A gene, in Dietzia sp. DQ12-45-1b, when the cells were grown in a medium containing C10, C12, and C14 n-alkanes [18]. Accordingly, Hoffmann et al. isolated 16 new P450 CYP153A genes from petroleum-contaminated soil and groundwater and divided them into three clusters (A, B, and C). Cluster A contained four new cytochrome P450 sequences, whose identity with P450balk at the amino acid level was 79–88%. Clusters B and C each contained six new P450 sequences, which were 71–73% and 52–55% identical to P450balk amino acid sequence, respectively [15].

In addition, Funhoff et al. showed that the heterologous expression of CYP153A6 can hydroxylate medium-chain- length alkanes (C6 to C11) to 1-alkanols. For the terminal carbon atom position, the maximal turnover number is 70 min− 1 and the regiospecificity is > 95% [11, 19].

Structures of CYP153A

The sequence homology of P450 enzymes from different families is very low, perhaps less than 20%. However, the overall protein structure is similar among the different P450s [20]. Generally, the conserved structures of CYPs are described as α A–L and β 1–10. The helix-rich secondary structure forms a heme-binding core, and the β-bulge location includes the thiolate heme ligand, referred to as the Cys-pocket. In addition, there is a structurally conserved loop between αK and the Cys-pocket, known as the ‘meander’ loop, that comprises 7–10 amino acid residues. It plays a role in heme-binding and stabilizing tertiary structures [21].

In addition to the conserved heme-binding region, the P450 enzyme has non-conserved regions acting as reductase interaction sites and substrate-binding regions. There are differences among CYP reductase interaction sites that lie between the heme-binding region αk/k’, the ‘meander’ loop, and Cys-pocket [22]. The non-conserved regions involved in substrate recognition and binding are described as substrate recognition sites (SRSs). SRS1 lies in the extraordinarily variable loop area between α B and α C (BC loop). SRS2 is positioned in the C-terminal of α F, while SRS3 and SRS4 span across the N-terminal areas of α G and α I. SRS5 contains the region from the conserved EXXR motif to the β1–4 strand. SRS6 is located between the β4 − 1 strand and β4 − 2 strand. The B-C loop (the loop between the B and C helices) and the F-G loop (the loop between the F and G helices) exhibit widespread differences. This structural variety confers considerable functional flexibility in P450s [21, 23].

CYP153A shares a conserved protein fold and heme-binding domain. The crystal structures of CYP153A from different microorganisms have been solved, revealing several structural features that contribute to its function and stability. CYP153A7(P450pyr from Sphingomonas sp. HXN-200, PDB: 3RWL)was the first reported CYP153A family three-dimensional structure, which explains its broad substrate range for hydroxylation. Owing to its high flexibility, researchers found that residues 88–96 in the B-C loop at the activity site were missing [24].

In contrast to P450BM3, P450pyr shows pronounced terminal selectivity. Functional sites have been engineered to produce mutants (P450pyrSM1) that catalyze subterminal hydroxylation. The binding pocket structure is responsible for the different substrate site selectivity and enantioselectivity. P450pyr has a compact binding pocket (7.1 Å in width) that makes the substrate position itself vertically, whereas the terminal C-H has the shortest distance (2.8 Å) and contains the heme O atom. By contrast, the P450pyrSM1 binding pocket is wider (10.3Å in width) because the F403I and L302V mutations disrupted the hydrophobic cluster. The wider pocket permits the substrate to exhibit a horizontal orientation relative to heme, leading to its preference for subterminal hydroxylation [25].

Kirton et al. resolved the three-dimensional structures of native CYP153AM.aq (CYP153A33 from Marinobacter aquaeolei, PDB: 5FYF) and CYP153AM.aq with ω-C12OH complexes (PDB: 5FYG) [26]. The complex structure suggested that the substrate tunnel geometry appeared as the tapering structure, and conferred the complex with high selectivity for the terminal carbon atom. In addition, CYP153AM.aq showed some notable characteristics in the F-helix, F/G loop, and B/C loop of its SRSs. The extended F-helix twisted the dodecanoic acid alkyl chain (13.3 Å) and shielded it from the F/G loop. The carboxyl group of dodecanoic acid is close to the amide side chains of Q129 and V141 on the B/C loop. According to the SRS structure, it can be assumed that there are no appropriate anchor sites for longer alkyl chains.

Fiorentini obtained 20 CYP153s, of which 17 represented single monooxygenase domains (hereafter named cand_1, cand_2, etc.) and the remaining three were multidomain proteins containing N-terminal CYP, FDR, and C-terminal FDX domains. Among these, the cand_1 (PDB: 6HQD), cand_10 (PDB: 6HQG), and cand_15 (PDB: 6HQW) structures were solved. The three CYP153 enzymes have different substrate preferences, with cand_1 being active in N-BOC-pyrrolidine and indigo, while cand_10 is active in octane. Furthermore, cand_15 is active in both N-BOC-pyrrolidine and octane. The researchers compared these three structures with CYP153A7 and CYP153A33 and found that conformational changes and sequence variations in the B/C-loop; F, G, and H helices; and F/G loop were the most obvious [27].

Gao, L et al. reported structural features of CYP153A from Mycobacterium sp. WY-01. It has high thermal stability, which is due in part to the presence of a disulfide bond that stabilizes the protein structure. This disulfide bond is formed between two cysteine residues located near the heme-binding site and is thought to contribute to the enzyme’s resistance to denaturation at high temperatures [28].

Molecular modifications of CYP153A

CYP153A is a P450 enzyme that requires a chaperone protein for catalysis. However, the specific type of chaperone protein utilized has an impact on the catalytic activity of CYP153A. Bordeaux et al. selected CYP153A13a from the Alcanivorax borkumensis SK2 strain as the catalytic domain and chose the P450RhF reductase site (RhFRed) - which includes FMN and NAD(P)H binding sites as well as [2Fe-2 S] ferredoxin center - obtained from Rhodococcus sp. strain to serve as the chaperone protein. Ultimately, these two enzymes were fused together and expressed in E. coli. The purified CYP153A13a-RhFRed was characterized by in vitro catalytic alkane hydroxylation with decane and octane as substrates. As only 1-decanol and 1-octanol were detected in the product, the fused CYP153A13a-RhFRed was shown to have very high chemical and regioselectivity for alkane catalysis [2].

Scheps et al. engineered a fusion protein consisting of CYP153AG307A from Marinobacter aquaeolei and the reductase domain of CPRBM3 from Bacillus megaterium, resulting in catalysis of 427 mg/L ω-OH12 production from 1 g/L dodecanoic acid (C12-FA). They generated a fusion protein, CYP153AM.aq-CPRBM3, by combining CYP153AG307A with the reductase of CYP116B3 from Rhodococcus ruber (Pfor), resulting in a 4-fold increase in conversion efficiency for the production of 105 mg/L ω-OH12 from 1 g/L dodecanoic acid (C12-FA) compared to that of CYP153AM.aq-CPRPfor [29]. Scheps et al. fused CYP153AP.sp from Polaromonas sp. strain JS666 with the reductase CPRBM3. In comparison with the wild-type CYP153AP.sp monooxygenase, the conversion efficiency of the fusion protein CYP153AP.sp-CPRBM3 for 1-butanol improved from 0.59 to 0.72 g/L [30].

At present, multiple three-dimensional structures of the CYP153A family have been resolved [24, 27]. Therefore, rational and semi-rational design strategies can be used to alternate the catalytic recreation or substrate specificity. Additionally, Scheps et al. performed site-specific mutations in the fusion protein CYP153AP.sp-CPRBM3. Compared to the fusion protein CYP153AP.sp-CPRBM3, the CYP153AP.sp (G254A)-CPRBM3 variation increased the conversion efficiency for the oxidation of n-butane to n-butanol from 0.72 to 2.99 g/L [30]. Seifert et al. determined the structure of CYP153A, obtained from Marinobacter aquaeolei VT8. The amino acids F87 and A328 residing in the substrate recognition site were considered mutation hot spots that altered chemical and regional selectivity. The double mutant F87A/A328I showed a dramatic increase in C2-hydroxylation regioselectivity (95%) [31].

Hauer et al. modified the fused protein CYP153AM.aq-CPRBM3 and showed that the protein exhibited enhanced terminal hydroxylation of fatty acids. Based on the homology in the structure of the CYP153AM.aq heme domain, modifications were performed using semi-rational mutagenesis. The selection residues were located in the substrate binding pocket and the substrate entry channel. The G307 position was located in the I-helix of the substrate binding pocket. The mutant G307A showed higher conservation for dodecanoic acid than the wild-type enzyme. Additionally, T302 was also located in the substrate binding pocket, whereas S233 was located in the substrate access channel. Compared with the wild-type enzyme, the triple mutant G307A/S233G/T302I showed a 2-fold increase in terminal hydroxylation efficiency for dodecanoic acid [32].

Jung et al. did site-specific saturation mutagenesis of CYP153A35 at the substrate binding channel to enhance the substrate binding affinity. The double mutant G297A/D131S showed an increase of 130% in the production of ω-hydroxylated palmitic acid in vivo compared with the wild-type enzyme and 42% compared with the D131S mutant [33].

Duan et al. paired putidaredoxin (Pdx) and putidaredoxin reductase (Pdr) of Pseudomonas putida as redox proteins with CYP153A of Marinobacter aquaeolei to form fusion proteins. CYP153A G307A had been reported to improve activities toward medium- and long-chain fatty acids [44]. Therefore, the fusion protein used CYP153A G307A mutant as a template, followed by selecting seven hydrophilic amino acid residues in the substrate binding pocket of CYP153A and 14 sites of the PDX interaction interface with CYP153A for engineering modification. Through site-directed saturation mutagenesis and two rounds of iterative saturation mutagenesis, a mutant (Variant II; G307A, S453N, S120R, P165N) was generated with significantly increased catalytic activity (33.0 ± 2.2 mg/L). S453N was located in the substrate binding pocket, and the mutants S120Y and P165N were located at the PDX interaction interface with CYP153A. Kinetics analyses of electron transfer from PDX to CYP153A showed that Variant II had the highest catalytic efficiency (5.16 × 106/min/M) and electron coupling efficiency (54.5% ± 9.7%). Thus, these findings suggest that the activity improvement for fusion protein variants resulted from enhanced electron transfer [34].

These molecular modifications have been successful in improving the properties of CYP153A, making it a more versatile and efficient enzyme for biotechnological applications.

Future directions and challenges

The emerging research areas and applications of CYP153A involve a wide range, such as the synthesis of pharmaceutical intermediates, biosensors, biodegradation of microplastics, and biocatalytic production of biodiesel, and in synthetic biology [13], CYP153A could be used as a building block in synthetic biology approaches to develop novel biosynthetic pathways and metabolic engineering strategies for the production of complex molecules. Applications of CYP153 enzymes consist of the conversion of terpene limonene into perillyl alcohol, a putative anticancer agent as properly as the functionality to hydroxylate piperidines, pyrrolidines, and azetidines to beneficial pharmaceutical intermediates [35]. CYP153A can be used as a biocatalyst to produce biodiesel from waste edible oil [36], which indicates that CYP153A may have potential application prospects in biodiesel production. In addition, CYP153A can also be synthetic bioplastic monomers [37], which indicates that CYP153A may have potential application prospects in the bioremediation of microplastic pollution.

While CYP153A has shown promising applications in bioremediation and biocatalysis, there are still limitations and challenges that need to be addressed. It is an oxygenase enzyme that requires oxygen for its catalytic activity, which may limit its use under certain conditions where oxygen availability is limited [38]. In spite of significant research efforts, the electron transport pathway between CYP153A and its reducing domain remains incompletely understood. This hinders the ability to engineer the enzyme for improved properties or fully exploit its potential in certain applications [21]. Finally, CYP153A has a significant substrate inhibition effect. For example, Lundemo et al. performed substrate inhibition experiments with dodecanoic acid (C12:0) and found that more 12-hydroxy dodecanoic acid (ω-OHC12:0) was obtained when the substrate was in the solid state compared to its liquid state. In the product inhibition experiment, they proved that when the concentration of 12-hydroxy dodecanoic acid increased from 1 to 5 mM, the product formation rate decreased from 0.3 mM/h to lower than 0.1 mM/h [39]. These studies suggest that substrate inhibition may be a potential limitation of CYP153A-mediated biotransformation reactions, and further research is needed to better understand the underlying mechanisms and to develop strategies to mitigate this effect.

In conclusion, CYP153A is a versatile cytochrome P450 enzyme that has been widely studied for its ability to catalyze a range of oxidation reactions on diverse substrates. While there are some limitations and challenges associated with studying and exploiting CYP153A, ongoing research is exploring new avenues for its application in synthetic biology, biocatalysis, and drug discovery. The potential of CYP153A to produce complex molecules and enable the development of novel biosynthetic pathways makes it an important target for future research in the fields of biotechnology and pharmaceuticals.