Search

The cytochrome P450 enzymes are ubiquitous heme-thiolate proteins performing regioselective and stereoselective oxygenation reactions in cellular metabolism. Due to their broad substrate scope and catalytic versatility, P450 enzymes are also attractive candidates for many industrial and biopharmaceutical applications. For particular uses, enzyme properties of P450s can be further optimized through directed evolution, rational, and semi-rational engineering approaches, all of which introduce mutations within the P450 structures. In this review, we describe the recent applications of these P450 engineering approaches and highlight the key regions and residues that have been identified using such approaches. These “hotspots” lie within critical functional areas of the P450 structure, including the active site, the substrate access channel, and the redox partner interaction interface. Keywords: Cytochrome P450, Protein engineering, Rational design, Crystal structure

Since 1970s, aplysiatoxins (ATXs), a class of biologically active dermatoxins, were identified from the marine mollusk Stylocheilus longicauda, whilst further research indicated that ATXs were originally metabolized by cyanobacteria. So far, there have been 45 aplysiatoxin derivatives discovered from marine cyanobacteria with various geographies. Recently, we isolated two neo-debromoaplysiatoxins, neo-debromoaplysiatoxin G (1) and neo-debromoaplysiatoxin H (2) from the cyanobacterium Lyngbya sp. collected from the South China Sea. The freeze-dried cyanobacterium was extracted with liquid–liquid extraction of organic solvents, and then was subjected to multiple chromatographies to yield neo-debromoaplysiatoxin G (1) (3.6 mg) and neo-debromoaplysiatoxin H (2) (4.3 mg). They were elucidated with spectroscopic methods. Moreover, the brine shrimp toxicity of the aplysiatoxin derivatives representing differential structural classifications indicated that the debromoaplysiatoxin was the most toxic compound (half inhibitory concentration (IC50) value = 0.34 ± 0.036 µM). While neo-aplysiatoxins (neo-ATXs) did not exhibit apparent brine shrimp toxicity, but showed potent blocking action against potassium channel Kv1.5, likewise, compounds 1 and 2 with IC50 values of 1.79 ± 0.22 µM and 1.46 ± 0.14 µM, respectively. Therefore, much of the current knowledge suggests the ATXs with different structure modifications may modulate multiple cellular signaling processes in animal systems leading to the harmful effects on public health.

Bacterial secondary metabolites have huge application potential in multiple industries. Biosynthesis of bacterial secondary metabolites are commonly encoded in a set of genes that are organized in the secondary metabolism biosynthetic gene clusters (SMBGCs). The development of genome sequencing technology facilitates mining bacterial SMBGCs. Marine Streptomyces is a valuable resource of bacterial secondary metabolites. In this study, 87 marine Streptomyces genomes were obtained and carried out into comparative genomic analysis, which revealed their high genetic diversity due to pan-genomes owning 123,302 orthologous clusters. Phylogenomic analysis indicated that the majority of Marine Streptomyces were classified into three clades named Clade I, II, and III, containing 23, 38, and 22 strains, respectively. Genomic annotations revealed that SMBGCs in the genomes of marine Streptomyces ranged from 16 to 84. Statistical analysis pointed out that phylotypes and ecotypes were both associated with SMBGCs distribution patterns. The Clade I and marine sediment-derived Streptomyces harbored more specific SMBGCs, which consisted of several common ones; whereas the Clade II and marine invertebrate-derived Streptomyces have more SMBGCs, acting as more plentiful resources for mining secondary metabolites. This study is beneficial for broadening our knowledge about SMBGC distribution patterns in marine Streptomyces and developing their secondary metabolites in the future.

Four biosurfactant-producing bacteria strains with high n-hexadecane degradation efficiency were isolated from activated sludge in a sequential batch reactor (SBR) in Weizhou terminal oilfield wastewater treatment plant, designated SBR14, SBR27, SBR28, and SBR45. Strains SBR14 and SBR45 were identified as Leclercia adecarboxylata, while strains SBR27 and SBR28 were identified as Enterobacter sp, based on 16S rDNA sequence analysis. The strains maintained growth activity under the following conditions: n-hexadecane concentration of 0.05-1% (v/v), salinity of 5-100 g/L, and pH of 5.0-9.0. Under optimal conditions: salinity of 15-25 g/L, pH of 6.0-7.0, inoculation amount of 5%, and a temperature and shaking speed of 37 °C and 160 rpm, respectively, the hexadecane (0.3%, v/v) degradation rate of each strain reached 93.74%, 65.66%, 73.27%, and 87.79% respectively, after 16 days of incubation. The crude products of these four strains were extracted, and the purified products were analyzed by thin layer chromatography and Fourier transform infrared spectroscopy. The products were identified as phospholipids biosurfactant, with yields of 0.564, 0.605, 0.435, and 0.657 g/L, for strains SBR14, SBR27, SBR28, and SBR45 respectively. The growth of strains agrees with the Logistic model, exhibiting specific growth rates of 0.1375, 0.254, 0.145, and 0.066 d-1, respectively. Meanwhile, hexadecane utilization followed the first order reaction kinetics model, with half value periods of 5.874, 10.046, 7.967, and 6.729 days, respectively. [ABSTRACT FROM AUTHOR]

In this study, the performance and microbial diversity of an oilfield wastewater treatment plant with an integrated anaerobic baffled reactor (ABR) and sequencing batch reactor (SBR) system were investigated. The analysis showed that the range of influent chemical oxygen demand and oil concentration was 215–731 and 9–52 mg/L, while the effluent concentration decreased to 30–87 and 2–8 mg/L after treatment, giving mean removal efficiencies of 88.5% and 85.0%, respectively. The removal efficiency of other indices in the wastewater (S2–, total suspended solids, and NH3 –N) reached 99.0%, 94.0%, and 80.1%, respectively. The microbial community analysis showed that the dominant bacterial and fungal species identified in the systems are halotolerant, suggesting that the process performances stated in this study were made possible by the adaptation of halotolerant microorganisms. Moreover, the results indicated that bacterial genera including Marinobacterium, Marinobacter, Thiomicrospira, Methylophaga, and Pseudomonas were likely to be actively involved in the decomposition processes of oil pollutants in the system. Furthermore, fungal communities were evenly distributed, with Ascomycota, Basidiomycota, and Zygomycota as the main phyla. Among them, Alternaria, Meyerozyma, Cryptococcus, Aspergillus, Candida, Stachybotrys, Fusarium, Blastobotrys, Mortierella, Rasamsonia, and Geminibasidium were abundant in the ABR and SBR systems, and may involve in the degradation process. [ABSTRACT FROM AUTHOR]

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol and a diagnostic tool for type C Niemann-Pick disease. Two position-specific P450 enzymes are involved in the post-polyketide modification of filipin during its biosynthesis, thereby providing molecular diversity to the "filipin complex." CYP105P1 and CYP105D6 from Streptomyces avermitilis, despite their high sequence similarities, catalyze filipin hydroxylation at different positions, C26 and C1', respectively. Here, we determined the crystal structure of the CYP105P1 filipin I complex. The distal pocket of CYP105P1 has the second largest size among P450 hydroxylases that act on macrolide substrates. Compared with previously determined substrate-free structures, the FG helices showed significant closing motion on substrate binding. The long BC loop region adopts a unique extended conformation without a B' helix. The binding site is essentially hydrophobic, but numerous water molecules are involved in recognizing the polyol side of the substrate. Therefore, the distal pocket of CYP105P1 provides a specific environment for the large filipin substrate to bind with its pro-S side of position C26 directed toward the heme iron. The ligand-free CYP105D6 structure was also determined. A small sub-pocket accommodating the long alkyl side chain of filipin I was observed in the CYP105P1 structure but was absent in the CYP105D6 structure, indicating that filipin cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation can explain the strict regiospecificity of these enzymes. [ABSTRACT FROM AUTHOR]

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol in biological membranes. The filipin biosynthetic pathway of Streptomyces avermitilis contains two position-specific hydroxylases, C26-specific CYP105P1 and C1'-specific CYP105D6. In this study, we describe the three X-ray crystal structures of CYP105P1: the ligand-free wild-type (WT-free), 4-phenylimidazole-bound wild-type (WT-4PI), and ligand-free H72A mutant (H72A-free) forms. The BC loop region in the WT-free structure has a unique feature; the side chain of His72 within this region is ligated to the heme iron. On the other hand, this region is highly disordered and widely open in WT-4PI and H72A-free structures, respectively. Histidine ligation of wild-type CYP105P1 was not detectable in solution, and a type II spectral change was clearly observed when 4-phenylimidazole was titrated. The H72A mutant showed spectroscopic characteristics that were almost identical to those of the wild-type protein. In the H72A-free structure, there is a large pocket that is of the same size as the filipin molecule. The highly flexible feature of the BC loop region of CYP105P1 may be required to accept a large hydrophobic substrate. [ABSTRACT FROM AUTHOR]

Cytochrome P450 enzymes are promising biocatalysts for industrial use because they catalyze site-selective C–H oxidation and have diverse catalytic reactions and a broad substrate range. In this study, the 2α-hydroxylation activity of CYP154C2 from Streptomyces avermitilis MA-4680T toward androstenedione (ASD) was identified by an in vitro conversion assay. The testosterone (TES)-bound structure of CYP154C2 was solved at 1.42 Å, and this structure was used to design eight mutants, including single, double, and triple mutants, to improve the conversion efficiency. Mutants L88F/ M191F and M191F/V285L were found to enhance the conversion rates significantly (i.e., 8.9-fold and 7.4-fold for TES, 46.5-fold and 19.5-fold for ASD, respectively) compared with the wild-type (WT) enzyme while retaining high 2α-position selectivity. The substrate binding affinity of the L88F/M191F mutant toward TES and ASD was enhanced compared with that of WT CYP154C2, supporting the measured increase in the conversion efficiencies. Moreover, the total turnover number and kcat/Km of the L88F/M191F and M191F/V285L mutants increased significantly. Interestingly, all mutants containing L88F generated 16α-hydroxylation products, suggesting that L88 in CYP154C2 plays a vital role in substrate selectivity and that the amino acid corresponding to L88 in the 154C subfamily affects the orientation of steroid binding and substrate selectivity. IMPORTANCE Hydroxylated derivatives of steroids play essential roles in medicine. Cytochrome P450 enzymes selectively hydroxylate methyne groups on steroids, which can dramatically change their polarity, biological activity and toxicity. There is a paucity of reports on the 2α-hydroxylation of steroids, and documented 2α-hydroxylate P450s show extremely low conversion efficiency and/or low regio- and stereoselectivity. This study conducted crystal structure analysis and structure-guided rational engineering of CYP154C2 and efficiently enhanced the conversion efficiency of TES and ASD with high regio- and stereoselectivity. Our results provide an effective strategy and theoretical basis for the 2α-hydroxylation of steroids, and the structure-guided rational design of P450s should facilitate P450 applications in the biosynthesis of steroid drugs. [ABSTRACT FROM AUTHOR]

Our previous study showed that CYP105D7, a substrate-promiscuous P450, catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, and compactin. In this study, 14 steroid compounds were screened using recombinant Escherichia coli cells harboring genes encoding CYP105D7 and redox partners (Pdx/Pdr, RhFRED, and FdxH/FprD), and the screening identified steroid A-ring 2β- and D-ring 16β-hydroxylation activity. Wild-type CYP105D7 was able to catalyze the hydroxylation of five steroids (testosterone, progesterone, 4-androstene-3,17-dione, adrenosterone, and cortisone) with low (<10%) conversion rates. Structure-guided site-directed mutagenesis of arginine residues around the substrate entrance and active site showed that the R70A and R190A single mutants and an R70A/R190A double mutant exhibited greatly enhanced conversion rates for steroid hydroxylation. For the conversion of testosterone in particular, the R70A/R190A mutant's kcat/Km values increased 1.35-fold and the in vivo conversion rates increased significantly by almost 9-fold with high regio- and stereoselectivity. Molecular docking analysis revealed that when Arg70 and Arg190 were replaced with alanine, the volume of the substrate access and binding pocket increased 1.08-fold, which might facilitate improvement of the hydroxylation efficiency of steroids. IMPORTANCE Cytochrome P450 monooxygenases (P450s) are able to introduce oxygen atoms into nonreactive hydrocarbon compounds under mild conditions, thereby offering significant advantages compared to chemical catalysts. Promiscuous P450s with broad substrate specificity and reaction diversity have significant potential for applications in various fields, including synthetic biology. The study of the function, molecular mechanisms, and rational engineering of substrate-promiscuous P450s from microbial sources is important to fulfill this potential. Here, we present a microbial substrate-promiscuous P450, CYP105D7, which can catalyze hydroxylation of steroids. The loss of the bulky side chains of Arg70 and Arg190 in the active site and substrate entrance resulted in an up to 9-fold increase in the substrate conversion rate. These findings will support future rational and semirational engineering of P450s for applications as biocatalysts. [ABSTRACT FROM AUTHOR]