Expanding the biocatalytic toolbox of flavoprotein monooxygenases from Rhodococcus jostii RHA1

With the aim to enlarge the set of available flavoprotein monooxygenases, we have cloned 8 unexplored genes from Rhodococcus jostii RHA1 that were predicted to encode class B flavoprotein monooxygenases. Each monooxygenase can be expressed as soluble protein and has been tested for conversion of sulfides and ketones. Not only enantioselective sulfoxidations, but also enantioselective Baeyer–Villiger oxidations could be performed with this set of monooxygenases. Interestingly, in contrast to known class B flavoprotein monooxygenases, all studied biocatalysts showed no nicotinamide coenzyme preference. This feature coincides with the fact that the respective sequences appear to form a discrete group of sequence related proteins, distinct from the known class B flavoprotein monooxygenases subclasses: the so-called flavin-containing monooxygenases (FMOs), N-hydroxylating monooxygenases (NMOs) and Type I Baeyer–Villiger monooxygenases (BVMOs). Taken together, these data reveal the existence of a new subclass of class B flavoprotein monooxygenases, which we coined as Type II FMOs, that can perform Baeyer–Villiger oxidations and accept both NADPH and NADH as coenzyme. The uncovered biocatalytic properties of the studied Type II FMOs make this newly recognized subclass of monooxygenases of potential interest for biocatalytic applications.     

Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening

To expand the available set of Baeyer-Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.     

Biocatalysis as an alternative for the production of chiral epoxides: A comparative review

Enantiopure epoxides are remarkably versatile intermediates for the synthesis of numerous biologically active targets, to which considerable efforts have been devoted either chemically or biologically during the past few decades. This review will emphasize the application of biocatalysis as an efficient alternative that complements conventional chemical reactions, with a special focus on the epoxidation reactions catalyzed with monooxygenases and chloroperoxidases and the hydrolytic kinetic resolution catalyzed with epoxide hydrolases. Their scopes and limitations will be elaborately discussed as compared with their chemical counterparts. These biocatalytic approaches have not only provided environmentally friendly alternatives, but also displayed advantages for certain types of enantiopure epoxides, and could serve as potential tools for synthetic chemists.     

Monooxygenation of aromatic compounds by flavin‐dependent monooxygenases

Many flavoenzymes catalyze hydroxylation of aromatic compounds especially phenolic compounds have been isolated and characterized. These enzymes can be classified as either single‐component or two‐component flavin‐dependent hydroxylases (monooxygenases). The hydroxylation reactions catalyzed by the enzymes in this group are useful for modifying the biological properties of phenolic compounds. This review aims to provide an in‐depth discussion of the current mechanistic understanding of representative flavin‐dependent monooxygenases including 3‐hydroxy‐benzoate 4‐hydroxylase (PHBH, a single‐component hydroxylase), 3‐hydroxyphenylacetate 4‐hydroxylase (HPAH, a two‐component hydroxylase), and other monooxygenases which catalyze reactions in addition to hydroxylation, including 2‐methyl‐3‐hydroxypyridine‐5‐carboxylate oxygenase (MHPCO, a single‐component enzyme that catalyzes aromatic‐ring cleavage), and HadA monooxygenase (a two‐component enzyme that catalyzes additional group elimination reaction). These enzymes have different unique structural features which dictate their reactivity toward various substrates and influence their ability to stabilize flavin intermediates such as C4a‐hydroperoxyflavin. Understanding the key catalytic residues and the active site environments important for governing enzyme reactivity will undoubtedly facilitate future work in enzyme engineering or enzyme redesign for the development of biocatalytic methods for the synthesis of valuable compounds.     

Biocatalytic Hydroxylations Catalyzed by Cytochromes P-450—Problems and Prospects

Due to their selectivity biocatalytic hydroxylations catalysed by monooxygenases or dioxygenases arouse considerable interest. The most studied and distributed type of monooxygenases are cytochromes P-450, which are a supergene family of proteins that catalyse the oxidation of lipophilic compounds through the insertion of 1 oxygen atom of O₂ into the substrate. For biocatalytic processes those cytochromes deserve attention, are involved in the catabolism of nutrients in microorganisms or in the metabolism of physiological substrates in higher eucaryotes. Mammalian cytochromes P-450, which catalyse the oxidation of xenobiotics (synthetic drugs, pesticides, hazardous waste compounds, are hardly suitable for this purpose. Processes with immobilized cytochromes P-450 can be excluded. The use of wild or genetically transformed strains of microorganisms is a suitable way.     

Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases

Flavoprotein monooxygenases are involved in a wide variety of biological processes including drug detoxification, biodegradation of aromatic compounds in the environment, biosynthesis of antibiotics and siderophores, and many others. The reactions use NAD(P)H and O2 as co-substrates and insert one atom of oxygen into the substrate. The flavin-dependent monooxygenases utilize a general cycle in which NAD(P)H reduces the flavin, and the reduced flavin reacts with O2 to form a C4a-(hydro)peroxyflavin intermediate, which is the oxygenating agent. This complicated catalytic process has diverse requirements that are difficult to be satisfied by a single site. Two general strategies have evolved to satisfy these requirements. para-Hydroxybenzoate hydroxylase, the paradigm for the single-component flavoprotein monooxygenases, is one of the most thoroughly studied of all enzymes. This enzyme undergoes significant protein and flavin dynamics during catalysis. There is an open conformation that gives access of substrate and product to solvent, and a closed or in conformation for the reaction with oxygen and the hydroxylation to occur. This closed form prevents solvent from destabilizing the hydroperoxyflavin intermediate. Finally, there is an out conformation achieved by movement of the isoalloxazine toward the solvent, which exposes its N5 for hydride delivery from NAD(P)H. The protein coordinates these dynamic events during catalysis. The second strategy uses a reductase to catalyze the reduction of the flavin and an oxygenase that uses the reduced flavin as a substrate to react with oxygen and hydroxylate the organic substrate. These two-component systems must be able to transfer reduced flavin from the reductase to the oxygenase and stabilize a C4a-peroxyflavin until a substrate binds to be hydroxylated, all before flavin oxidation and release of H2O2. Again, protein dynamics are important for catalytic success.     

Mapping the substrate binding site of phenylacetone monooxygenase from Thermobifida fusca by mutational analysis

Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.     

Discovery of a thermostable Baeyer–Villiger monooxygenase by genome mining

Baeyer–Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer–Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (k_cat = 1.9 s^–1, K_M = 59 M). The enzyme exhibits moderate enantioselectivity with -methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer–Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52°C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.     

Tools and ingredients for the biocatalytic synthesis of metabolites

Metabolic networks have been an interesting starting point not only for the design of synthetic routes in a similar sequence of reactions, e.g., in biomimetic syntheses, but also for assembling a number of biocatalytic steps by preparing the required enzymes and auxiliary reagents. Retrosynthetic analysis involving multiple biocatalytic reactions steps therefore needs to consider the practically realized biocatalytic single steps. The opportunities for route selection are enlarged if novel synthetic reactions connecting easily available starting materials and products are found, and/or both biocatalytic and classical reactions of organic chemistry are utilized. Tools and ingredients for biocatalytic synthesis are of special interest for reactions difficult to achieve by classical organic synthesis. Densely and differentially functionalized small molecules do not allow much space for protecting or activating groups. Biocatalytic reactions have therefore performed well for a number of useful metabolites in enantiopure form to achieve full functionality. Although many well-known metabolites from classical biochemistry have only been prepared in racemic form, it is of fundamental interest to have these available in enantiomerically pure form. Biocatalytic reactions with nature's privileged chiral catalysts appear to be a promising synthetic strategy towards these metabolites, especially when sensitive or stable-isotope-labeled metabolites are to be prepared. The main applications for these metabolites are as references materials in metabolomics, as enzyme substrates for the characterization of metabolic enzyme activities and as potential pharmaceuticals in biomedical research. The use of stable-isotope-labeled metabolites can thereby simplify in vivo applications and metabolic flux analyses.     

X‐ray crystal structure of cytochrome P450 monooxygenase CYP101J2 from Sphingobium yanoikuyae strain B2

The cytochrome P450 monooxygenases (P450s) catalyze a vast array of oxygenation reactions that can be useful in biocatalytic applications. CYP101J2 from Sphingobium yanoikuyae is a P450 that catalyzes the hydroxylation of 1,8‐cineole. Here we report the crystallization and X‐ray structure elucidation of recombinant CYP101J2 to 1.8 Å resolution. The CYP101J2 structure shows the canonical P450‐fold and has an open conformation in the absence of substrate. Analysis of the structure revealed that CYP101J2, in the absence of substrate, forms a well‐ordered substrate‐binding channel that suggests a unique form of substrate guidance in comparison to other bacterial 1,8‐cineole‐hydroxylating P450 enzymes. Proteins 2017; 85:945–950. © 2016 Wiley Periodicals, Inc.     

Biocatalysis--key to sustainable industrial chemistry

The ongoing trends to process improvements, cost reductions and increasing quality, safety, health and environment requirements of industrial chemical transformations have strengthened the translation of global biocatalysis research work into industrial applications. One focus has been on biocatalytic single-step reactions with one or two substrates, the identification of bottlenecks and molecular as well as engineering approaches to overcome these bottlenecks. Robust industrial procedures have been established along classes of biocatalytic single-step reactions. Multi-step reactions and multi-component reactions (MCRs) enable a bottom-up approach with biocatalytic reactions working together in one compartment and recations hindering each other within different compartments or steps. The understanding of the catalytic functions of known and new enzymes is key for the development of new sustainable chemical transformations.     

Flavoprotein oxidases: classification and applications

This review provides an overview of oxidases that utilise a flavin cofactor for catalysis. This class of oxidative flavoenzymes has shown to harbour a large number of biotechnologically interesting enzymes. Applications range from their use as biocatalysts for the synthesis of pharmaceutical compounds to the integration in biosensors. Through the recent developments in genome sequencing, the number of newly discovered oxidases is steadily growing. Recent progress in the field of flavoprotein oxidase discovery and the obtained biochemical knowledge on these enzymes are reviewed. Except for a structure-based classification of known flavoprotein oxidases, also their potential in recent biotechnological applications is discussed.     

The stereospecificity of bacterial external flavoprotein monooxygenases for nicotinamide adenine dinucleotide.

It is known that orcinol hydroxylase shows A-stereospecificity for nicotinamide adenine dinucleotide when the enzyme reaction involves the true substrate, orcinol, but when the reaction is carried out in the presence of pseudosubstrates, this enzyme shows an isotope dependent mixed-type Stereospecificity, the degree of which depends on the uncoupling activity of the employed pseudosubstrate [Ribbons, D. W., Ohta, Y. and Higgins, I. J. (1972) in Miami Winter Symposium, The Molecular Basis of Electron Transport (Schultz, J., and Cameron, B. R., eds.), Vol. 4, pp. 251–274, Academic Press, New York]. In this report, the nicotinamide nucleotide stereospecificity of other external flavoprotein monooxygenases from bacterial sources is presented. All of the monooxygenases examined, namely, melilotate hydroxylase, m-hydroxybenzoate-6-hydroxylase, resorcinol hydroxylase, and salicylate hydroxylase, invariably show A-stereospecificity, as in the case of orcinol hydroxylase with true substrate. Unlike orcinol hydroxylase, however, the stereospecificity of salicylate hydroxylase remains A-stereospecific even with pure pseudosubstrate (i.e., benzoate) regardless of the position of the deuterium at the dihydronicotinamide 4-position. On the basis of the information obtained in this investigation, a general rule pertaining to the stereospecificity of nicotinamide nucleotide enzymes is proposed that all external flavoprotein monooxygenases have A-stereospecificity with respect to nicotinamide adenine dinucleotide.     

Cellular distribution, metabolism and regulation of the xanthine oxidoreductase enzyme system

Xanthine oxidase (EC 1.1.3.22) and xanthine dehydrogenase (EC 1.1.1. 204) are both members of the molybdenum hydroxylase flavoprotein family and represent different forms of the same gene product. The two enzyme forms and their reactions are often referred to as xanthine oxidoreductase (XOR) activity. Physiologically, XOR is known as the rate-limiting enzyme in purine catabolism but has also been shown to be able to metabolize a number of other physiological compounds. Recent studies have also demonstrated its ability to metabolize xenobiotics, including a number of anticancer compounds, to their active metabolites. During the past 10 years, evidence has mounted to support a role for XOR in the pathophysiology of inflammatory diseases and atherosclerosis as well as its previously determined role in ischemia-reperfusion injury. While significant progress has recently been made in our understanding of the physiological and biochemical nature of this enzyme system, considerable work still needs to be done. This paper will review some of the more recent work characterizing the interactions and the factors that influence the interactions of XOR with various physiological and xenobiotic compounds.     

Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His₁₀-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.The incorporation of one atom of oxygen during hydroxylation, epoxidation, sulfoxidation, or Baeyer-Villiger oxidation is a common initial step of the aerobic degradation of aromatic compounds by microorganisms. In bacteria, these reactions are most frequently catalyzed by inducible flavoprotein monooxygenases (EC 1.14.13 [57]). The majority of these enzymes (so-called single-component flavoprotein monooxygenases) utilize electrons from NAD(P)H, which are transferred to a non-covalently bound flavin adenine dinucleotide (FAD) in order to activate molecular oxygen as a flavin (hydro)peroxide. Depending on the protonation of this intermediate and the type of substrate, an oxygen atom is then incorporated by nucleophilic or electrophilic attack. More recently, different two-component flavoprotein monooxygenases have been characterized (57). These systems cover an NAD(P)H-dependent flavin reductase in order to generate reduced flavin and an oxygenase that utilizes this cofactor for the activation of oxygen.The exquisite regio- and stereoselectivities of oxygen insertion by flavoprotein monooxygenases favor these enzymes for biocatalytic applications (23, 24, 33). This is especially true because chemical synthesis approaches by hetero- or homogenic catalysis often do not yield a sufficiently high enantiomeric excess for the production of pharmaceuticals and their chiral building blocks. The use of oxygen as an inexpensive nontoxic oxidant and mild reaction conditions are additional advantages with the potential for increasing the environmental sustainability of oxygenase-catalyzed biotransformations.The necessity for expensive cofactors is perhaps the most striking drawback limiting the industrial application of flavoprotein monooxygenases. Different electrochemical and enzymatic procedures for in vitro cofactor regeneration are available (20, 21, 32, 52, 56), but these systems are currently lacking long-term stability. As a consequence, the practical application of flavoprotein monooxygenases is virtually restricted to in vivo systems in which cofactor regeneration is mediated by the metabolism of the expression host (45, 49). The limitations of whole-cell-mediated biotransformations by substrate and/or product toxicity can be overcome by means of two-phase systems, as was recently shown for the two-component styrene monooxygenase (SMO) from Pseudomonas sp. strain VLB120 (45).Two-component flavoprotein monooxygenases present additional challenges for biocatalytic applications. The need for two separate protein components may hamper attempts at recombinant enzyme expression, the application of immobilized enzymes in cell-free systems, and the detection of novel oxygenases during activity-based metagenome-screening approaches. Moreover, and as was already shown for the two-component SMO from Pseudomonas sp. strain VLB120 (44), the interprotein transfer of reduced FAD is accompanied to a certain extent by the auto-oxidative formation of reactive oxygen species such as hydrogen peroxide (Fig. ​(Fig.1).1). Auto-oxidation of reduced FAD not only decreases the efficiency of the oxygenation process but also negatively interferes with the physiological conditions of the cell. The extent of oxidative stress is considerably increased when FAD oxidoreductase activity exceeds oxygenase activity and uncoupling becomes dominant.Open in a separate windowFIG. 1.Catalytic mechanism of two-component SMOs and the formation of oxidative stress by uncoupling between FAD oxidoreductase (StyB) and oxygenase (StyA) (adapted from reference 36). FAD_OX and FAD_RED, oxidized and reduced forms of FAD, respectively.Presently the details of reduced-FAD transfer between the oxygenase and FAD oxidoreductase components of SMOs are not known. Recent kinetic studies have indicated that reduced FAD is transferred by a mixed mechanism in which direct contact of both proteins and free diffusion of the reduced cofactor play a role (25). This hypothesis is supported by the work of Otto and coworkers in which the formation of hydrogen peroxide was shown to be reciprocally proportional to the concentration of active oxygenase StyA (44). The high level of efficiency of self-sufficient cytochrome P450 enzymes compared to that of multicomponent types is attributed to the closer location between the heme-containing P450 domain (oxygenase) and a reductase domain (FAD/flavin mononucleotide [FMN] and NADH binding site), which should also promote the efficiency of diffusive transfer (38). These self-sufficient P450 systems are of high biocatalytic interest (8, 34, 39), and it is likely that other types of self-sufficient monooxygenases (e.g., flavoenzymes) behave in a similar way.Members of the gram-positive genus Rhodococcus are characterized by their exceptionally high level of metabolic versatility toward a broad range of organic substrates (31). Large genome sizes, the presence of megaplasmids, and a distinct gene redundancy (multiple enzyme homologs) favor these organisms as a promising source for novel enzymes (59). Moreover, several studies have provided evidence of functionally convergent evolution of the catabolic activities of rhodococci and proteobacteria (12, 37). Since most research on bacterial catabolic activities has so far been performed on the latter group, novel enzymes and mechanisms are likely to be identified in gram-positive bacteria.The nocardioform actinobacterium Rhodococcus opacus 1CP was originally isolated from contaminated soil by enrichment with 4-chloro- and 2,4-dichlorophenol as the sole carbon sources (18). To solve questions related to the enzymes involved in chlorophenol mineralization, an R. opacus 1CP clone library was generated, leading to the identification of a 9-kb genomic fragment harboring genes with a presumed function in styrene metabolism. Sequence analysis indicated the presence of an open reading frame (ORF) encoding a fusion protein composed of an oxygenase and a reductase subunit with a high level of similarity to the corresponding subunits of two-component SMOs from pseudomonads. Recombinant expression and biochemical characterization confirmed that it has enantioselective styrene epoxidation ability and showed that StyA2B is the first representative of a new class of NADH- and flavin-dependent single-component flavoprotein monooxygenases.     

Chemoenzymatic and microbial dynamic kinetic resolutions

This review tracks a decade of dynamic kinetic resolution developments with a biocatalytic inclination using enzymatic/microbial means for the resolution part followed by the racemization reactions either by means of enzymatic or chemocatalyst. These fast developments are due to the ability of the biocatalysts to significantly reduce the number of synthetic steps which are common for conventional synthesis. Future developments in novel reactions and products of dynamic kinetic resolutions should consider factors that are needed to be extracted at the early synthetic stage to avoid inhibition at scale-up stage have been highlighted.     

Site-Directed Mutagenesis of Cytochrome P450scc. II. Effect of Replacement of the Arg425 and Arg426 Residues on the Structural and Functional Properties of the Cytochrome P450scc

Cytochrome P450-dependent monooxygenases, in spite of their wide distribution, can be simply divided into a few groups differing in the location of the electron transfer chain and their composition. The two main groups of cytochrome P450-dependent monooxygenases are the mitochondrial and microsomal enzymes. While in two-component microsomal cytochrome P450-dependent monooxygenases electrons are supplied to cytochrome P450 by a flavoprotein (NADPH-cytochrome P450 reductase), in three-component mitochondrial monooxygenases the electrons are supplied to cytochrome P450 by a low molecular weight protein (ferredoxin). The interaction of cytochrome P450 with NADPH-cytochrome P450 reductase and ferredoxin is the subject of intensive studies. Using chemical modification, chemical cross-linking, and sitedirected mutagenesis, we identified surface exposed positively charged residues of cytochrome P450scc which might be important for interaction with adrenodoxin. Theoretical analysis of the distribution of surface electrostatic potential in cytochrome P450 indicates that in contrast to microsomal monooxygenases, cytochromes P450 of mitochondrial type, and cholesterol side-chain cleavage cytochrome P450 (P450scc) in part, carry on the proximal surface an evidently positively charged site that is formed by residues Arg425 and Arg426. In the present work, to estimate the functional role of Arg425 and Arg426 of cytochrome P450scc, we used site-directed mutagenesis to replace these residues with glutamine. The results indicate that residues Arg425 and Arg426 are involved in the formation of a heme-binding center and electrostatic interaction of cytochrome P450scc with its physiological electron-transfer partner, adrenodoxin.     

Exploring the structural basis of substrate preferences in Baeyer-Villiger monooxygenases: insight from steroid monooxygenase

Steroid monooxygenase (STMO) from Rhodococcus rhodochrous catalyzes the Baeyer-Villiger conversion of progesterone into progesterone acetate using FAD as prosthetic group and NADPH as reducing cofactor. The enzyme shares high sequence similarity with well characterized Baeyer-Villiger monooxygenases, including phenylacetone monooxygenase and cyclohexanone monooxygenase. The comparative biochemical and structural analysis of STMO can be particularly insightful with regard to the understanding of the substrate-specificity properties of Baeyer-Villiger monooxygenases that are emerging as promising tools in biocatalytic applications and as targets for prodrug activation. The crystal structures of STMO in the native, NADP(+)-bound, and two mutant forms reveal structural details on this microbial steroid-degrading enzyme. The binding of the nicotinamide ring of NADP(+) is shifted with respect to the flavin compared with that observed in other monooxygenases of the same class. This finding fully supports the idea that NADP(H) adopts various positions during the catalytic cycle to perform its multiple functions in catalysis. The active site closely resembles that of phenylacetone monooxygenase. This observation led us to discover that STMO is capable of acting also on phenylacetone, which implies an impressive level of substrate promiscuity. The investigation of six mutants that target residues on the surface of the substrate-binding site reveals that enzymatic conversions of both progesterone and phenylacetone are largely insensitive to relatively drastic amino acid changes, with some mutants even displaying enhanced activity on progesterone. These features possibly reflect the fact that these enzymes are continuously evolving to acquire new activities, depending on the emerging availabilities of new compounds in the living environment.     

Mechanistic and structural studies of the N-hydroxylating flavoprotein monooxygenases

The N-hydroxylating flavoprotein monooxygenases are siderophore biosynthetic enzymes that catalyze the hydroxylation of the sidechain amino-group of ornithine or lysine or the primary amino-group of putrescine. This hydroxylated product is subsequently formylated or acylated and incorporated into the siderophore. Importantly, the modified amino-group is a hydroxamate and serves as an iron chelating moiety in the siderophore. This review describes recent work to characterize the ornithine hydroxylases from Pseudomonas aeruginosa (PvdA) and Aspergillus fumigatus (SidA) and the lysine hydroxylase from Escherichia coli (IucD). This includes summaries of steady and transient state kinetic data for all three enzymes and the X-ray crystallographic structure of PvdA.     

Extending the capabilities of nature's most versatile catalysts: directed evolution of mammalian xenobiotic-metabolizing P450s

Cytochrome P450 enzymes are amongst the most versatile enzymatic catalysts known. The ability to introduce a single atom of oxygen into an organic substrate has led to the diversification and exploitation of these enzymes throughout nature. Nowhere is this versatility more apparent than in the mammalian liver, where P450 monooxygenases catalyze the metabolic clearance of innumerate drugs and other environmental chemicals. In addition to the aromatic and aliphatic hydroxylations, N- and O-dealkylations, and heteroatom oxidations that are common in drug metabolism, many more unusual reactions catalyzed by P450s have been discovered, including reductions, group transfers and other biotransformations not typically associated with monooxygenases. A research area that shows great potential for development over the next few decades is the directed evolution of P450s as biocatalysts. Mammalian xenobiotic-metabolizing P450s are especially well suited to such protein engineering due to their ability to interact with relatively wide ranges of substrates with marked differences in structure and physicochemical properties. Typical characteristics, such as the low turnover rates and poor coupling seen during the metabolism of xenobiotics, as well as the enzyme specificity towards particular substrates and reactions, can be improved by directed evolution. This mini-review will cover the fundamental enabling technologies required to successfully engineer P450s, examine the work done to date on the directed evolution of mammalian forms, and provide a perspective on what will be required for the successful implementation of engineered enzymes.