Bacterial Cytochrome P450 Research Articles

Engineering of Microbial Substrate Promiscuous CYP105A5 for Improving the Flavonoid Hydroxylation

Bacterial cytochrome P450 (CYP) enzymes are versatile biocatalysts that are responsible for the biotransformation of diverse endogenous substances. CYP105A5 from Streptomyces sp. showed substrate flexibility with different flavonoids and was able to catalyze O-demethylation of biochanin A, regioselective C3′-hydroxylation of daidzein, genistein, and naringenin, and additional C8-hydroxylation for daidzein using heterologous redox partners putidaredoxin and putidaredoxin reductase. By rational design of substrate-binding pocket based on experimental data, homology modeling, and molecular docking analysis, we enhanced the product formation rate of flavonoids. The double mutant L100A/I302A and L100A/I408N exhibited greatly enhanced in vivo conversion rates for flavonoid hydroxylation. Particularly, the L100A/I302A mutant’s kcat/Km values and in vivo conversion rate increased by 1.68-fold and 2.57-fold, respectively, for naringenin. Overall, our result might facilitate the potential use of CYP105A5 for future modification and application in whole-cell biocatalysts for the production of valuable polyphenols.

Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity.

Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the β subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.

Weit verbreitete bifunktionelle, bakterielle Cytochrom P450 Enzyme katalysieren sowohl eine intramolekulare C−C‐Bindung incyclo‐l‐Tyr‐l‐Tyr als auch dessen Verknüpfung mit Nukleinbasen

AbstractModifizierende Enzyme spielen eine wichtige Rolle in der Biosynthese von Naturstoffen. Wir berichten hier über sechs orthologe Gencluster mit jeweils zwei Genen für die Biosynthese von Mycocyclosin und Guatyromycinen. Die Expression der CyclodipeptidsynthasegenegymA1–gymA6inEscherichia coliführte zur Produktion voncyclo‐l‐Tyr‐l‐Tyr als Hauptprodukt. Durch Rekonstruktion des Biosynthesewegs inStreptomyces albusin Kombination mit biochemischen Untersuchungen konnte bewiesen werden, dass die Cytochrom P450 Enzyme sowohl als intramolekulare Oxidasen, wie auch als Nukleinbasen‐Transferasen agieren. Sie katalysieren nicht nur die oxidative C−C Verknüpfung innerhalb voncyclo‐l‐Tyr‐l‐Tyr, woraus Mycocyclosin resultiert, sondern auch dessen Verbindung mit Guanin und Hypoxanthin. Somit sind sie verantwortlich für die Entstehung der tyrosinhaltigen Guatyromycine, anstatt der bisher bekannten tryptophanhaltigen Nukleinbasen‐Addukte. Phylogenetische Daten lassen vermuten, dass es mindestens 47 solcher orthologen GymBs gibt. Dies spricht für eine weitverbreitete Enzymklasse.

Widely Distributed Bifunctional Bacterial Cytochrome P450 Enzymes Catalyze both Intramolecular C−C Bond Formation incyclo‐l‐Tyr‐l‐Tyr and Its Coupling with Nucleobases

Tailoring enzymes are important modification biocatalysts in natural product biosynthesis. We report herein six orthologous two‐gene clusters for mycocyclosin and guatyromycine biosynthesis. Expression of the cyclodipeptide synthase genes gymA1 –gymA6 in Escherichia coli resulted in the formation of cyclo‐l‐Tyr‐l‐Tyr as the major product. Reconstruction of the biosynthetic pathways in Streptomyces albus and biochemical investigation proved that the cytochrome P450 enzymes GymB1–GymB6 act as both intramolecular oxidases and intermolecular nucleobase transferases. They catalyze not only the oxidative C−C coupling within cyclo‐l‐Tyr‐l‐Tyr, leading to mycocyclosin, but also its connection with guanine and hypoxanthine, and are thus responsible for the formation of tyrosine‐containing guatyromycines, instead of the reported tryptophan‐nucleobase adducts. Phylogenetic data suggest the presence of at least 47 GymB orthologues, indicating the occurrence of a widely distributed enzyme class.

Heterologous expression of bacterial cytochrome P450 from Microbacterium keratanolyticum ZY and its application in dichloromethane dechlorination

Dichloromethane (DCM) is a volatile halogenated hydrocarbon with teratogenic, mutagenic and carcinogenic effects. Biodegradation is generally regarded as an effective and economical approach of pollutant disposal. In this study, a novel strain was isolated and its cytochrome P450 was heterologously expressed for DCM degradation. The isolate, Microbacterium keratanolyticum ZY, was characterized as a Gram-positive, rod-shaped and flagella-existed bacterium without spores (GenBank No. SUB8814364; CCTCC M 2019953). After successive whole-genome sequencing, assembly and annotation, eight identified functional genes (encoding cytochrome P450, monooxygenase, dehalogenase and hydrolase) were successfully cloned and expressed in Escherichia coli BL21 (DE3). The recombinant strain expressing cytochrome P450 presented the highest degradation efficiency (90.6%). Moreover, the specific activity of the recombinant cytochrome P450 was more than 1.2 times that of the recombinant dehalogenase (from Methylobacterium rhodesianum H13) under their optimum conditions. The kinetics of DCM degradation by recombinant cytochrome P450 was well fitted with the Haldane model and the value of maximum specific degradation rate was determined to be 0.7 s−1. The DCM degradation might occur through successive hydroxylation, dehydrohalogenation, dechlorination and oxidation to generate gem-halohydrin, formyl chloride, formaldehyde and formic acid. The study helps to comprehensively understand the DCM dechlorination process under the actions of bacterial functional enzymes (cytochrome P450 and dehalogenase).

Uncovering of cytochrome P450 anatomy by SecStrAnnotator

Protein structural families are groups of homologous proteins defined by the organization of secondary structure elements (SSEs). Nowadays, many families contain vast numbers of structures, and the SSEs can help to orient within them. Communities around specific protein families have even developed specialized SSE annotations, always assigning the same name to the equivalent SSEs in homologous proteins. A detailed analysis of the groups of equivalent SSEs provides an overview of the studied family and enriches the analysis of any particular protein at hand. We developed a workflow for the analysis of the secondary structure anatomy of a protein family. We applied this analysis to the model family of cytochromes P450 (CYPs)—a family of important biotransformation enzymes with a community-wide used SSE annotation. We report the occurrence, typical length and amino acid sequence for the equivalent SSE groups, the conservation/variability of these properties and relationship to the substrate recognition sites. We also suggest a generic residue numbering scheme for the CYP family. Comparing the bacterial and eukaryotic part of the family highlights the significant differences and reveals a well-known anomalous group of bacterial CYPs with some typically eukaryotic features. Our workflow for SSE annotation for CYP and other families can be freely used at address https://sestra.ncbr.muni.cz.

Identification of Fungal Limonene-3-Hydroxylase for Biotechnological Menthol Production.

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as a medical component. So far, only extraction from plant material and chemical synthesis are possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies have already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs, or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low compared to that of the highly selective L3H enzymes from the biosynthetic pathway for menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered additional fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production, and its additional ability to convert α- and β-pinene to verbenol and pinocarveol, respectively.IMPORTANCE (-)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (-)-menthol production from renewable (+)-limonene.

An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

Cytochrome P450 (CYP) heme monooxygenases require two electrons for their catalytic cycle. For mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). No structure of a mammalian CYP–CPR complex has been solved experimentally, hindering understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage to the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP–CPR ET rates are low compared to those of soluble bacterial CYPs.

Kinetic Evidence for an Induced Fit Mechanism in the Binding of the Substrate Camphor by Cytochrome P450cam.

Bacterial cytochrome P450 (P450) 101A1 (P450cam) has served as a prototype among the P450 enzymes and has high catalytic activity towards its cognate substrate, camphor. X-ray crystallography and NMR and IR spectroscopy have demonstrated the existence of multiple conformations of many P450s, including P450cam. Kinetic studies have indicated that substrate binding to several P450s is dominated by a conformational selection process, in which the substrate binds an individual conformer(s) of the unliganded enzyme. P450cam was found to differ in that binding of the substrate camphor is dominated by an induced fit mechanism, in which the enzyme binds camphor and then changes conformation, as evidenced by the equivalence of binding eigenvalues observed when varying both camphor and P450cam concentrations. The accessory protein putidaredoxin had no effect on substrate binding. Estimation of the rate of dissociation of the P450cam·camphor complex (15 s-1) and fitting of the data yield a minimal kinetic mechanism in which camphor binds (1.5 × 107 M-1 s-1) and the initial P450cam•camphor complex undergoes a reversible equilibrium (k forward 112 s-1, k reverse 28 s-1) to a final complex. This induced fit mechanism differs from those reported for several mammalian P450s and bacterial P450BM-3, indicative of the diversity of how P450s recognize multiple substrates. However, similar behavior was not observed with the alternate substrates (+)-α-pinene and 2-adamantanone, which probably utilize a conformational selection process.

Oxidative Diversification of Steroids by Nature-Inspired Scanning Glycine Mutagenesis of P450BM3 (CYP102A1)

Steroidal compounds are some of the most prescribed medicines, being indicated for the treatment of a variety of conditions including inflammation, heart disease, and cancer. Synthetic approaches to functionalized steroids are important for generating steroidal agents for drug screening and development. However, chemical activation is challenging because of the predominance of inert, aliphatic C–H bonds in steroids. Here, we report the engineering of the stable, highly active bacterial cytochrome P450 enzyme P450BM3 (CYP102A1) from Bacillus megaterium for the mono- and dihydroxylation of androstenedione (AD), dehydroepiandrosterone (DHEA), and testosterone (TST). In order to design altered steroid binding orientations, we compared the structure of wild type P450BM3 with the steroid C19-demethylase CYP19A1 with AD bound within its active site and identified regions of the I helix and the β4 strand that blocked this binding orientation in P450BM3. Scanning glycine mutagenesis across 11 residues in these two regions led to steroid oxidation products not previously reported for P450BM3. Combining these glycine mutations in a second round of mutagenesis led to a small library of P450BM3 variants capable of selective (up to 97%) oxidation of AD, DHEA, and TST at the widest range of positions (C1, C2, C6, C7, C15, and C16) by a bacterial P450 enzyme. Computational docking of these steroids into molecular dynamics simulated structures of selective P450BM3 variants suggested crucial roles of glycine mutations in enabling different binding orientations from the wild type, including one that closely resembled that of AD in CYP19A1, while other mutations fine-tuned the product selectivity. This approach of designing mutations by taking inspiration from nature can be applied to other substrates and enzymes for the synthesis of natural products and their derivatives.

Resonance Raman spectroscopic studies of peroxo and hydroperoxo intermediates in lauric acid (LA)-bound cytochrome P450 119

Cytochromes P450 bind and cleave dioxygen to generate a potent intermediate compound I, capable of hydroxylating inert hydrocarbon substrates. Cytochrome P450 119, a bacterial cytochrome P450 that serves as a good model system for the study of the intermediate states in the P450 catalytic cycle. CYP119 is found in high temperature and sulfur rich environments. Though the natural substrate and redox partner are still unknown, a potential application of such thermophilic P450s is utilizing them as biocatalysts in biotechnological industry; e.g., the synthesis of organic compounds otherwise requiring hostile environments like extremes of pH or temperature. In the present work the oxygenated complex of this enzyme bound to lauric acid, a surrogate substrate known to have a good binding affinity, was studied by a combination of cryoradiolysis and resonance Raman spectroscopy, to trap and characterize active site structures of the key fleeting enzymatic intermediates, including the peroxo and hydroperoxo species.

Riboflavin Is Directly Involved in N-Dealkylation Catalyzed by Bacterial Cytochrome P450 Monooxygenases.

Like a vast number of enzymes in nature, bacterial cytochrome P450 monooxygenases require an activated form of flavin as a cofactor for catalytic activity. Riboflavin is the precursor of FAD and FMN that serves as indispensable cofactor for flavoenzymes. In contrast to previous notions, herein we describe the identification of an electron-transfer process that is directly mediated by riboflavin for N-dealkylation by bacterial P450 monooxygenases. The electron relay from NADPH to riboflavin and then via activated oxygen to heme was proposed based on a combination of X-ray crystallography, molecular modeling and molecular dynamics simulation, site-directed mutagenesis and biochemical analysis of representative bacterial P450 monooxygenases. This study provides new insights into the electron transfer mechanism in bacterial P450 enzyme catalysis and likely in yeasts, fungi, plants and mammals.

Heterologous caffeic acid biosynthesis in Escherichia coli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450

BackgroundCaffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented l-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far.ResultsThe herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 µM or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even a virtually unproductive Cytochrome P450/redox system combination productive.ConclusionsThe caffeic acid titer achieved in this study is about 10% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without l-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.

Two‐step Screening for Identification of Drug‐metabolizing Bacterial Cytochromes P450 with Diversified Selectivity

AbstractThe evaluation of drug metabolites is compulsory during drug development. Since recently, bacterial cytochromes P450 and their mutated variants have attracted considerable interest as an alternative to hepatic P450s for the synthesis of human drug metabolites. Thus, straightforward screening approaches are required that enable rapid identification and evaluation of drug‐metabolizing bacterial P450s with different product selectivities. Herein, we report a two‐step screening method for discovery and characterization of new P450s from actinomycetes that enable oxidation of various drugs. In the first step, substrate profiling with three structurally different model drugs, ritonavir, testosterone, amitriptyline, allowed us to select CYP105D and CYP107Z from Streptomyces platensis DSM 40041 that accepted all model substrates and produced human‐like drug metabolites. In the second step, activity tests with an array of 25 structurally‐related molecules and derivatives of the three model compounds revealed a correlation between structural variations in the target drugs and the enzyme chemo‐ and regioselectivity.

Rearrangement-Free Hydroxylation of Methylcubanes by a Cytochrome P450: The Case for Dynamical Coupling of C-H Abstraction and Rebound.

The highly strained cubylmethyl radical undergoes one of the fastest radical rearrangements known (reported k = 2.9 × 1010 s-1 at 25 °C) through scission of two bonds of the cube. The rearrangement has previously been used as a mechanistic probe to detect radical-based pathways in enzyme-catalyzed C-H oxidations. This paper reports the discovery of highly selective cytochrome P450-catalyzed methylcubane oxidations which notionally proceed via cubylmethyl radical intermediates yet are remarkably free of rearrangement. The bacterial cytochrome P450 CYP101B1 from Novosphingobium aromaticivorans DSM 12444 is found to hydroxylate the methyl group of a range of methylcubane substrates containing a regio-directing carbonyl functionality at C-4. Unlike other reported P450-catalyzed methylcubane oxidations, the designed methylcubanes are hydroxylated with high efficiency and selectivity, giving cubylmethanols in yields of up to 93%. The lack of cubane core ring-opening implies that the cubylmethyl radicals formed during these CYP101B1-catalyzed hydroxylations must have very short lifetimes, of just a few picoseconds, which are too short for them to manifest the side reactivity characteristic of a fully equilibrated P450 intermediate. We propose that the apparent ultrafast radical rebound can be explained by a mechanism in which C-H abstraction and C-O bond formation are merged into a dynamically coupled process, effectively bypassing a discrete radical intermediate. Related dynamical phenomena can be proposed to predict how P450s may achieve various other modes of reactivity by controlling the formation and fate of radical intermediates. In principle, dynamical ideas and two-state reactivity are each individually able to explain apparent ultrashort radical lifetimes in P450 catalysis, but they are best considered together.

Crystal structure of bacterial CYP116B5 heme domain: New insights on class VII P450s structural flexibility and peroxygenase activity

Class VII cytochromes P450 are self-sufficient enzymes carrying a phthalate family oxygenase-like reductase domain and a P450 domain fused in a single polypeptide chain. The biocatalytic applications of CYP116B members are limited by the need of the NADPH cofactor and the lack of crystal structures as a starting point for protein engineering. Nevertheless, we demonstrated that the heme domain of CYP116B5 can use hydrogen peroxide as electron donor bypassing the need of NADPH.Here, we report the crystal structure of CYP116B5 heme domain in complex with histidine at 2.6 Å of resolution. The structure reveals the typical P450 fold and a closed conformation with an active site cavity of 284 Å3 in volume, accommodating a histidine molecule forming a hydrogen bond with the water molecule present as 6th heme iron ligand. MD simulations in the absence of any ligand revealed the opening of a tunnel connecting the active site to the protein surface through the movement of F-, G- and H-helices.A structural alignment with bacterial cytochromes P450 allowed the identification of amino acids in the proximal heme site potentially involved in peroxygenase activity.The availability of the crystal structure provides the bases for the structure-guided design of new biocatalysts.

Evaluation of Luminogenic Substrates as Probe Substrates for Bacterial Cytochrome P450 Enzymes: Application to Mycobacterium tuberculosis

Several cytochrome P450 enzymes (CYPs) encoded in the genome of Mycobacterium tuberculosis (Mtb) are considered potential new drug targets due to the essential roles they play in bacterial viability and in the establishment of chronic intracellular infection. Identification of inhibitors of Mtb CYPs at present is conducted by ultraviolet-visible (UV-vis) optical titration experiments or by metabolism studies using endogenous substrates, such as cholesterol and lanosterol. The first technique requires high enzyme concentrations and volumes, while analysis of steroid hydroxylation is dependent on low-throughput analytical methods. Luciferin-based luminogenic substrates have proven to be very sensitive substrates for the high-throughput profiling of inhibitors of human CYPs. In the present study, 17 pro-luciferins were evaluated as substrates for Mtb CYP121A1, CYP124A1, CYP125A1, CYP130A1, and CYP142A1. Luciferin-BE was identified as an excellent probe substrate for CYP130A1, resulting in a high luminescence yield after addition of luciferase and adenosine triphosphate (ATP). Its applicability for high-throughput screening was supported by a high Z’-factor and high signal-to-background ratio. Using this substrate, the inhibitory properties of a selection of known inhibitors could be characterized using significantly less protein concentration when compared to UV-vis optical titration experiments. Although several luminogenic substrates were also identified for CYP121A1, CYP124A1, CYP125A1, and CYP142A1, their relatively low yield of luminescence and low signal-to-background ratios make them less suitable for high-throughput screening since high enzyme concentrations will be needed. Further structural optimization of luminogenic substrates will be necessary to obtain more sensitive probe substrates for these Mtb CYPs.

Influence of organic amendments used for benz[a]anthracene remediation in a farmland soil: pollutant distribution and bacterial changes

Organic amendments are usually carried out at field-scale for efficient remediation of organic pollutants; however, their effects on pollutant distribution and the corresponding microbial mechanisms were rarely discussed. The main aim of this study was to compare the fate of benz[a]anthracene in soil amended by several bioremediation materials and underlying microbial mechanisms. In this study, the potential for biotransformation of polycyclic aromatic hydrocarbon in a farmland soil was investigated in microcosms spiked with 14C-benz[a]anthracene as the tracer. A series of organic amendments including lignin, straw, mushroom culture waste, and cow manure, as well as a fungal inoculum of Pleurotus ostreatus, were compared. Illumina sequencing was introduced to reveal the bacterial community in different amendments. The metagenomic function was predicted with the bioinformatics tool of phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt). From the results, the lignin-contained substrates (lignin, straw, mushroom culture waste) showed increase trend in the dissipation of benz[a]anthracene, while Pleurotus ostreatus and cow manure resulted in opposite trends. Specifically, mushroom culture waste mainly increased 14C to the formation of humin-bound residue (39.5 ± 6.8%); lignin amendment significantly (P < 0.05) enhanced the mineralization to CO2 (7.38 ± 0.89%) and humic acid–related nonextractable residue (9.77 ± 0.45%). The influence of straw on the environmental fate of benz[a]anthracene was marginal. High-throughput sequencing of 16S rRNA genes demonstrated that mushroom culture waste and lignin significantly changed bacterial community composition, leading to increases in the relative abundance of Pseudomonadaceae, Methylophilaceae, Bacillaceae, and Burkholderiaceae. Moreover, the result of PICRUSt showed that the genes-encoding bacterial cytochrome P450 enzymes were significantly increased in the lignin treatment, suggesting a possible co-metabolism between lignin degradation and PAH mineralization. These findings suggest lignin-containing organic amendments could be promising soil remediation agents of benz[a]anthracene by stimulating mineralization and sequestration of pollutants.

Structural analysis of Cytochrome P450 BM3 mutant M11 in complex with dithiothreitol.

The bacterial Cytochrome P450 (CYP) BM3 (CYP102A1) is one of the most active CYP isoforms. BM3 mutants can serve as a model for human drug-metabolizing CYPs and/or as biocatalyst for selective formation of drug metabolites. Hence, molecular and computational biologists have in the last two decades shown strong interest in the discovery and design of novel BM3 variants with optimized activity and selectivity for substrate conversion. This led e.g. to the discovery of mutant M11 that is able to metabolize a variety of drugs and drug-like compounds with relatively high activity. In order to further improve our understanding of CYP binding and reactions, we performed a co-crystallization study of mutant M11 and report here the three-dimensional structure M11 in complex with dithiothreitol (DTT) at a resolution of 2.16 Å. The structure shows that DTT can coordinate to the Fe atom in the heme group. UV/Vis spectroscopy and molecular dynamics simulation studies underline this finding and as first structure of the CYP BM3 mutant M11 in complex with a ligand, it offers a basis for structure-based design of novel mutants.

Biomimetic and Biocatalytic Synthesis of Bruceol

AbstractThe first total synthesis of bruceol has been achieved using a biomimetic cascade cyclization initiated by a stereoselective Jacobsen–Katsuki epoxidation (and kinetic resolution) of racemic protobruceol‐I. A bacterial cytochrome P450 monooxygenase was also found to catalyze the conversion of protobruceol‐I into bruceol. The first full analysis of the NMR data of natural bruceol suggested that “isobruceol” was a previously unrecognized natural product also isolated from Philotheca brucei. This was confirmed by the re‐isolation, synthesis, and X‐ray analysis of isobruceol. In total, eight stereoisomers and structural isomers of bruceol have been synthesized in a highly divergent approach.