A flavoprotein monooxygenase that catalyses a Baeyer-Villiger reaction and thioether oxidation using NADH as the nicotinamide cofactor

A gene from the marine bacterium Stenotrophomonas maltophilia encodes a 38.6 kDa FAD‐containing flavoprotein (Uniprot B2FLR2) named S. maltophilia flavin‐containing monooxygenase (SMFMO), which catalyses the oxidation of thioethers and also the regioselective Baeyer–Villiger oxidation of the model substrate bicyclo[3.2.0]hept‐2‐en‐6‐one. The enzyme was unusual in its ability to employ either NADH or NADPH as nicotinamide cofactor. The K_M and k_cat values for NADH were 23.7±9.1 μM and 0.029 s^?1 and 27.3±5.3 μM and 0.022 s^?1 for NADPH. However, k_cat/K_M value for the ketone substrate in the presence of 100 μM cofactor was 17 times greater for NADH than for NADPH. SMFMO catalysed the quantitative conversion of 5 mM ketone in the presence of substoichiometric concentrations of NADH with the formate dehydrogenase cofactor recycling system, to give the 2‐oxa and 3‐oxa lactone products of Baeyer–Villiger reaction in a ratio of 5:1, albeit with poor enantioselectivity. The conversion with NADPH was 15 %. SMFMO also catalysed the NADH‐dependent transformation of prochiral aromatic thioethers, giving in the best case, 80 % ee for the transformation of p‐chlorophenyl methyl sulfide to its R enantiomer. The structure of SMFMO reveals that the relaxation in cofactor specificity appears to be accomplished by the substitution of an arginine residue, responsible for recognition of the 2′‐phosphate on the NADPH ribose in related NADPH‐dependent FMOs, with a glutamine residue in SMFMO. SMFMO is thus representative of a separate class of single‐component, flavoprotein monooxygenases that catalyse NADH‐dependent oxidations from which possible sequences and strategies for developing NADH‐dependent biocatalysts for asymmetric oxygenation reactions might be identified.     

Controlling the Regioselectivity of Baeyer–Villiger Monooxygenases by Mutation of Active‐Site Residues

Baeyer–Villiger monooxygenase (BVMO)‐mediated regiodivergent conversions of asymmetric ketones can lead to the formation of “normal” or “abnormal” lactones. In a previous study, we were able to change the regioselectivity of a BVMO by mutation of the active‐site residues to smaller amino acids, which thus created more space. In this study, we demonstrate that this method can also be used for other BVMO/substrate combinations. We investigated the regioselectivity of 2‐oxo‐Δ³‐4,5,5‐trimethylcyclopentenylacetyl‐CoA monooxygenase from Pseudomonas putida (OTEMO) for cis‐bicyclo[3.2.0]hept‐2‐en‐6‐one ( 1 ) and trans‐dihydrocarvone ( 2 ), and we were able to switch the regioselectivity of this enzyme for one of the substrate enantiomers. The OTEMO wild‐type enzyme converted (?)‐ 1 into an equal (50:50) mixture of the normal and abnormal products. The F255A/F443V variant produced 90 % of the normal product, whereas the W501V variant formed up to 98 % of the abnormal product. OTEMO F255A exclusively produced the normal lactone from (+)‐ 2 , whereas the wild‐type enzyme was selective for the production of the abnormal product. The positions of these amino acids were equivalent to those mutated in the cyclohexanone monooxygenases from Arthrobacter sp. and Acinetobacter sp. (CHMO_Arthro and CHMO_Acineto) to switch their regioselectivity towards (+)‐ 2 , which suggests that there are hot spots in the active site of BVMOs that can be targeted with the aim to change the regioselectivity.     

Direct Access to Medium‐Chain α,ω‐Dicarboxylic Acids by Using a Baeyer–Villiger Monooxygenase of Abnormal Regioselectivity

Baeyer–Villiger monooxygenases (BVMOs) are versatile biocatalysts in organic synthesis that can generate esters or lactones by inserting a single oxygen atom adjacent to a carbonyl moiety. The regioselectivity of BVMOs is essential in determining the ratio of two regioisomers for converting asymmetric ketones. Herein, we report a novel BVMO from Pseudomonas aeruginosa (PaBVMO); this has been exploited for the direct synthesis of medium‐chain α,ω‐dicarboxylic acids through a Baeyer–Villiger oxidation–hydrolysis cascade. PaBVMO displayed the highest abnormal regioselectivity toward a variety of long‐chain aliphatic keto acids (C₁₆–C₂₀) to date, affording dicarboxylic monoesters with a ratio of up to 95 %. Upon chemical hydrolysis, α,ω‐dicarboxylic acids and fatty alcohols are readily obtained without further treatment; this significantly reduces the synthetic steps of α,ω‐dicarboxylic acids from renewable oils and fats.     

The Evolution of an Amine Dehydrogenase Biocatalyst for the Asymmetric Production of Chiral Amines

The reductive amination of ketones to produce chiral amines is an important transformation in the production of pharmaceutical intermediates. Therefore, industrially applicable enzymatic methods that enable the selective synthesis of chiral amines could be very useful. Using a phenylalanine dehydrogenase scaffold devoid of amine dehydrogenase activity, a robust amine dehydrogenase has been evolved with a single two‐site library allowing for the direct production of (R)‐1‐(4‐fluorophenyl)‐propyl‐2‐amine from para‐fluorophenylacetone with a k_cat value of 6.85 s⁻¹ and a K_M value of 7.75 mM for the ketone substrate. This is the first example of a highly active amine dehydrogenase capable of accepting aliphatic and benzylic ketone substrates. The stereoselectivity of the evolved amine dehydrogenase was very high (>99.8% ee) showing that high selectivity of the wild‐type phenylalanine dehydrogenase was conserved in the evolution process. When paired with glucose/glucose dehydrogenase, NADH cofactor can be effficiently regenerated and the reaction driven to over 93% conversion. The broad specificity, high selectivity, and near complete conversion render this amine dehydrogenase an attractive target for further evolution toward pharmaceutical compounds and subsequent application.     

In Vitro Double Oxidation of n‐Heptane with Direct Cofactor Regeneration

A novel concept for the direct oxidation of cycloalkanes to the corresponding cyclic ketones in a one‐pot synthesis in water with molecular oxygen as sole oxidizing agent was reported recently. Based on this concept we have developed a new strategy for the double oxidation of n‐heptane to enable a biocatalytic resolution for the direct synthesis of heptanone and (R)‐heptanols in a one‐pot reaction. The bicatalytic cascade employs an NADH driven P450 BM3 monooxygenase variant (WT^NADH, 19A12^NADH or CM1^NADH) and an (S)‐enantioselective alcohol dehydrogenase (RE‐ADH). In the initial step n‐heptane is hydroxylated under consumption of NADH to produce (R/S)‐heptanol. In the second oxidation step the (S)‐heptanol enantiomers are transformed to the corresponding ketones, reducing and thereby regenerating the cofactor. Characterization of initial hydroxylation step revealed high turnover frequencies (TOF) of up to 600 min⁻¹, as well as high coupling efficiencies using NADH as cofactor (up to 44%). In the cascade reaction a nearly 2‐fold improved product formation was achieved, compared to the single hydroxylation reaction. The total product concentration reached 1.1 mM, corresponding to a total turnover number (TTN) of 2500. Implementation of an additional cofactor regeneration system (D ‐glucose/glucose dehydrogenase) enabled a further enhancement in product formation with a total product concentration of 1.8 mM and a TTN of 3500.     

Crosslinked Aggregates of Fusion Enzymes in Microaqueous Organic Media

Baeyer-Villiger monooxygenases (BVMOs) are attractive for selectively oxidizing various ketones using oxygen into valuable esters and lactones. However, the application of BVMOs is restrained by cofactor dependency and enzyme instability combined with water-related downsides such as low substrate loading, low oxygen capacity, and water-induced side reactions. Herein, we described a redox-neutral linear cascade with in-situ cofactor regeneration catalyzed by fused alcohol dehydrogenase and cyclohexanone monooxygenase in aqueous and microaqueous organic media. The cascade conditions have been optimized regarding substrate concentrations as well as the amounts of enzymes and cofactors with the Design of Experiments (DoE). The carrier-free immobilization technique, crosslinked enzyme aggregates (CLEAs), was applied to fusion enzymes. The resultant fusion CLEAs were proven to function in microaqueous organic systems, in which the enzyme ratios, water contents (0.5–5 vol. %), and stability have been systematically studied. The fusion CLEAs showed promising operational (up to 5 cycles) and storage stability.     

Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A

The NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductaseenzyme is a required component in some novel biosynthetic vitaminC production processes. This enzyme catalyzes the conversionof 2,5-DKG to 2-keto-L-gulonic acid, which is an immediate precursorto L-ascorbic acid. Forty unique site-directed mutations weremade at five residues in the cofactor-binding pocket of 2,5-DKGreductase A in an attempt to improve its ability to use NADHas a cofactor. NADH is more stable, less expensive and moreprevalent in the cell than is NADPH. To the best of our knowledge,this is the first focused attempt to alter the cofactor specificityof a member of the aldo–keto reductase superfamily byengineering improved activity with NADH into the enzyme. Activityof the mutants with NADH or NADPH was assayed using activity-stainednative polyacrylamide gels. Eight of the mutants at three differentsites were identified as having improved activity with NADH.These mutants were purified and subjected to a kinetic characterizationwith NADH as a cofactor. The best mutant obtained, R238H, producedan almost 7-fold improvement in catalysis with NADH comparedwith the wild-type enzyme. Surprisingly, most of this catalyticimprovement appeared to be due to an improvement in the apparentk_cat for the reaction rather than a large improvement in theaffinity of the enzyme for NADH.     

Photochemically Induced Oxidative and Reductive Regeneration of NAD(P)+/NAD(P)H Cofactors: Applications in Biotransformations

Photosensitized regeneration of NAD(P)H cofactors is accomplished by biocatalyzed and artificially catalyzed transformations in photochemical assemblies. Photogenerated N,N'-dimethyl-4,4′-bipyridinium radical cation, MV^+., acts as electron carrier for the reduction of NADPH in the presence of the enzyme ferredoxin reductase and for the reduction of NADH in the presence of lipoamide dehydrogenase. For the photogeneration of MV^+. and subsequent NADPH formation, three different photosensitizers are applied: Ru(bpz)²₃⁺, Ru(bpy)²₃⁺, and Zn—TMPyP⁴⁺. The highest quantum yield for NADPH formation is observed with Ru(bpz)₃²⁺ and is ϕ = 1.7 × 10⁻¹. For NADH regeneration only Zn-TMPyP⁴⁺ can be applied. Ru(bpy)₃²⁺ and Ru(bpz)²⁺₃ interact with NADH in their excited or oxidized forms and therefore cannot be used as light-active compounds in the system. The NADPH regeneration cycle has been coupled to the biocatalyzed synthesis of glutamic acid. Although Ru(bpz)₃²⁺ is 42.5-fold more efficient than Ru(bpy)₃²⁺ in the regeneration of NADPH, the synthesis of glutamic acid is improved only by a factor of 2 in the presence of Ru(bpz)₃²⁺, implying that the coupled process is rate limiting. Oxidative regeneration of the NAD⁺ cofactor is accomplished in a photosystem that includes Ru(bpy)₃²⁺ as photosensitizer. The photoprocess is coupled to dehydrogenation of ethanol, propanol, lactic acid, and alanine with concomitant H₂ evolution. A photosystem that includes Ru(bpy)₃²⁺ as photosensitizer, ascorbate as electron donor, and chloro-tris-(3-diphenylphosphinobenzene sulfonate)Rh(I), RhCl(dpm)₃^3-, is catalytically active in the photoinduced regeneration of NAD(P)H cofactors. Mechanistic investigations show that photogenerated Ru(bpy)₃⁺ mediates the generation of a hydrido-rhodium complex that acts as a charge relay for the production of NAD(P)H.     

Redesign of the coenzyme specificity in L-Lactate dehydrogenase from Bacillus stearothermophilus using site-directed mutagenesis and media engineering

L-Lactate dehydrogenase (LDH) from Bacillus stearothermophilusis a redox enzyme which has a strong preference for NADH overNADPH as coenzyme. To exclude NADPH from the coenzyme-bindingpocket, LDH contains a conserved aspartate residue at position52. However, this residue is probably not solely responsiblefor the NADH specificity. In this report we examine the possibilitiesof altering the coenzyme specificity of LDH by introducing arange of different point mutations in the coenzyme-binding domain.Furthermore, after choosing the mutant with the highest selectivityfor NADPH, we also investigated the possibility of further alteringthe coenzyme specificity by adding an organic solvent to thereaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-foldincreased specificity to NADPH over the wild-type LDH in a reactionmixture containing 15% methanol. Furthermore, the NADPH turnovernumber of this mutant was increased almost fourfold as comparedwith wild-type LDH. To explain the altered coenzyme specificityexhibited by the D52SI51K double mutant, molecular dynamicssimulations were performed.     

An Alcohol Dehydrogenase from the Short-Chain Dehydrogenase/Reductase Family of Enzymes for the Lactonization of Hexane-1,6-diol

Biocatalytic production of lactones, and in particular ϵ-caprolactone (CL), have gained increasing interest as a greener route to polymer building blocks, especially through the use of Baeyer–Villiger monooxygenases (BVMOs). Despite several advances in the field, BVMOs, however, still suffer several practical limitations. Alcohol dehydrogenase (ADH)-mediated lactonization of diols in turn has received far less attention and very few enzymes have been identified for the conversion of diols to lactones, with horse-liver ADH (HLADH) remaining the catalyst of choice. Screening of a diverse panel of ADHs, AaSDR-1, a member of the short-chain dehydrogenase/reductase family, was found to produce ϵ-caprolactone from hexane-1,6-diol. Moreover, cofactor regeneration by an NADH oxidase eliminated the requirement of co-substrates, yielding water as the sole by-product. Despite lower turnover frequencies as compared to HLADH, higher selectivity was found for the production of CL, with HLADH forming significant amounts of 6-hydroxyhexanoic acid and adipic acid through aldehyde dehydrogenation/oxidation of the gem-diol intermediates. Also, CL yield were shown to be dependent on buffer choice, as structural elucidation of a Tris adduct confirmed the buffer amine to react with aliphatic aldehydes forming a Schiff-base intermediate which through further ADH oxidation, forms a tricyclic acetal product.     

Cofactor Regeneration of NAD+ from NADH: Novel Water‐Forming NADH Oxidases

Dehydrogenases with their superb enantioselectivity can be employed advantageously to prepare enantiomerically pure alcohols, hydroxy acids, and amino acids. For economic syntheses, however, the co‐substrate of dehydrogenases, the NAD(P)(H) cofactor, has to be regenerated. Whereas the problem of regenerating NADH from NAD⁺ can be considered solved, the inverse problem of regenerating NAD⁺ from NADH still awaits a definitive and practical solution. A possible solution is the oxidation of NADH to NAD⁺ with concomitant reduction of oxygen catalyzed by NADH oxidase (E.C. 1.6.‐.‐) which can reduce O₂ either to undesirable H₂O₂ or to innocuous H₂O. We have found and cloned two novel genes from Borrelia burgdorferi and Lactobacillus sanfranciscensis with hitherto only machine‐annotated NADH oxidase function. We have overexpressed the corresponding proteins and could prove the annotated function to be correct. As demonstrated with a more sensitive assay than employed previously, the two novel NADH oxidases reduce O₂ to H₂O.     

Improving the Enantioselectivity of CHMOBrevi1 for Asymmetric Synthesis of Podophyllotoxin Precursor

(R)-β-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called “focused rational iterative site-specific mutagenesis” (FRISM) at residues ranging from 6 Å from substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-β-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.     

Investigation of a New Type I Baeyer–Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability

Baeyer–Villiger monooxygenases (BVMOs) are remarkable biocatalysts, but, due to their low stability, their application in industry is hampered. Thus, there is a high demand to expand on the diversity and increase the stability of this class of enzyme. Starting from a known thermostable BVMO sequence from Thermocrispum municipale (TmCHMO), a novel BVMO from Amycolaptosis thermoflava (BVMO_Flava), which was successfully expressed in Escherichia coli BL21(DE3), was identified. The activity and stability of the purified enzyme was investigated and the substrate profile for structurally different cyclohexanones and cyclobutanones was assigned. The enzyme showed a lower activity than that of cyclohexanone monooxygenase (CHMO_Acineto) from Acinetobacter sp., as the prototype BVMO, but indicated higher kinetic stability by showing a twofold longer half-life at 30 °C. The thermodynamic stability, as represented by the melting temperature, resulted in a T_m value of 53.1 °C for BVMO_Flava, which was comparable to the T_m of TmCHMO (ΔT_m=1 °C) and significantly higher than the T_m value for CHMO_Acineto ((ΔT_m=14.6 °C)). A strong deviation between the thermodynamic and kinetic stabilities of BVMO_Flava was observed; this might have a major impact on future enzyme discovery for BVMOs and their synthetic applications.     

Cofactor engineering in cyanobacteria to overcome imbalance between NADPH and NADH: A mini review

Cyanobacteria can produce useful renewable fuels and high-value chemicals using sunlight and atmospheric carbon dioxide by photosynthesis. Genetic manipulation has increased the variety of chemicals that cyanobacteria can produce. However, their uniquely abundant NADPH-pool, in other words insufficient supply of NADH, tends to limit their production yields in case of utilizing NADH-dependent enzyme, which is quite common in heterotrophic microbes. To overcome this cofactor imbalance and enhance cyanobacterial fuel and chemical production, various approaches for cofactor engineering have been employed. In this review, we focus on three approaches: (1) utilization of NADPH-dependent enzymes, (2) increasing NADH production, and (3) changing cofactor specificity of NADH-dependent enzymes from NADH to NADPH.

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Discovery and Engineering of a Novel Baeyer-Villiger Monooxygenase with High Normal Regioselectivity

Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products (“normal” and “abnormal”) when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (k_cat/K_M) 8.9-fold to 484 s⁻¹ mM⁻¹ for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMO_C308L as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L⁻¹ d⁻¹. This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils.     

Engineering a Formate Dehydrogenase for NADPH Regeneration**

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD⁺-dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP⁺-utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD⁺ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (k_cat/K_m). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.     

A partial convergence in action of methylene blue and artemisinins: antagonism with chloroquine, a reversal with verapamil, and an insight into the antimalarial activity of chloroquine

Artemisinins rapidly oxidize leucomethylene blue (LMB) to methylene blue (MB); they also oxidize dihydroflavins such as the reduced conjugates RFH₂ of riboflavin (RF), and FADH₂ of the cofactor flavin adenine dinucleotide (FAD), to the corresponding flavins. Like the artemisinins, MB oxidizes FADH₂, but unlike artemisinins, it also oxidizes NAD(P)H. Like MB, artemisinins are implicated in the perturbation of redox balance in the malaria parasite by interfering with parasite flavoenzyme disulfide reductases. The oxidation of LMB by artemisinin is inhibited by chloroquine (CQ), an inhibition that is abruptly reversed by verapamil (VP). CQ also inhibits artemisinin‐mediated oxidation of RFH₂ generated from N‐benzyl‐1,4‐dihydronicotinamide (BNAH)–RF, or FADH₂ generated from NADPH or NADPH–Fre, an effect that is also modulated by verapamil. The inhibition likely proceeds by the association of LMB or dihydroflavin with CQ, possibly involving donor–acceptor or π complexes that hinder oxidation by artemisinin. VP competitively associates with CQ, liberating LMB or dihydroflavin from their respective CQ complexes. The observations explain the antagonism between CQ–MB and CQ–artemisinins in vitro, and are reconcilable with CQ perturbing intraparasitic redox homeostasis. They further suggest that a VP–CQ complex is a means by which VP reverses CQ resistance, wherein such a complex is not accessible to the putative CQ‐resistance transporter (PfCRT).     

Stereo-Divergent Enzyme Cascades to Convert Racemic 4-Phenyl-2-Butanol into either (S)- or (R)-Corresponding Chiral Amine

The synthesis of enantiopure chiral amines from racemic alcohols is a key transformation in the chemical industry, e. g., in the production of active pharmaceutical ingredients (APIs). However, this reaction remains challenging. In this work, we propose a one-pot enzymatic cascade for the direct conversion of a racemic alcohol into either (S)- or (R)-enantiomers of the corresponding amine, with in-situ cofactor recycling. This enzymatic cascade consists of two enantio-complementary alcohol dehydrogenases, both NADH and NADPH oxidase for in-situ recycling of NAD(P)⁺ cofactors, and either (S)- or (R)-enantioselective transaminase. This cell-free biocatalytic system has been successfully applied to the conversion of racemic 4-phenyl-2-butanol into the high value (S)- or (R)-enantiomers of the amine reaching good (73 % (S)) and excellent (>99 % (R)) enantioselectivities.     

Towards practical Baeyer-Villiger-monooxygenases: design of cyclohexanone monooxygenase mutants with enhanced oxidative stability

Baeyer–Villiger monooxygenases (BVMOs) catalyze the conversion of ketones and cyclic ketones into esters and lactones, respectively. Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is known to show an impressive substrate scope as well as exquisite chemo‐, regio‐, and enantioselectivity in many cases. Large‐scale synthetic applications of CHMO are hampered, however, by the instability of the enzyme. Oxidation of cysteine and methionine residues contributes to this instability. Designed mutations of all the methionine and cysteine residues in the CHMO wild type (WT) showed that the amino acids labile towards oxidation are mostly either surface‐exposed or located within the active site, whereas the two methionine residues identified for thermostabilization are buried within the folded protein. Combinatorial mutations gave rise to two stabilized mutants with either oxidative or thermal stability, without compromising the activity or stereoselectivity of the enzyme. The most oxidatively stabilized mutant retained nearly 40 % of its activity after incubation with H₂O₂ (0.2 M ), whereas the wild‐type enzyme's activity was completely abolished at concentrations as low as 5 mM H₂O₂. We propose that oxidation‐stable mutants might well be a “prerequisite” for thermostabilization, because laboratory‐evolved thermostability in CHMO might be masked by a high degree of oxidation instability.     

Characterization of a Self-Sufficient Cytochrome P450 Monooxygenase from Deinococcus apachensis for Enantioselective Benzylic Hydroxylation

A self-sufficient cytochrome P450 monooxygenase from Deinococcus apachensis (P450DA) was identified and successfully overexpressed in Escherichia coli BL21(DE3). P450DA would be a member of the CYP102D subfamily and assigned as CYP102D2 according to the phylogenetic tree and sequence alignment. Purification and characterization of the recombinant P450DA indicated both NADH and NADPH could be used by P450DA as a reducing cofactor. The recombinant E. coli (P450DA) strain was functionally active, showing excellent enantioselectivity for benzylic hydroxylation of methyl 2-phenylacetate. Further substrate scope studies revealed that P450DA is able to catalyze benzylic hydroxylation of a variety of compounds, affording the corresponding chiral benzylic alcohols in 86–99 % ee and 130–1020 total turnover numbers.