Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase

Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO_Acineto); this used NADH 4200‐fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen‐bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.     

A New Type of Stereoselectivity in Baeyer–Villiger Reactions: Access to E‐ and Z‐Olefins

A new concept for accessing configurationally defined trisubstituted olefins has been developed. Starting from a common ketone precursor of the type 4‐ethylidenecyclohexanone, Baeyer–Villiger monooxygenases are employed as catalysts in diastereoselective Baeyer–Villiger reactions leading to the corresponding E‐ or Z‐configurated lactones. Wild‐type cyclohexanone monooxygenase (CHMO) as catalyst delivers the E‐isomers and a directed evolution mutant the opposite Z‐isomers. Subsequent transition metal‐catalyzed chemical transformations of a key product containing a vinyl bromide moiety provide a variety of different trisubstituted E‐ or Z‐olefins. A model based on QM/MM sheds light on the origin of this unusual type of diastereoselectivity. In contrast to this biocatalytic approach, traditional Baeyer–Villiger reagents such as m‐CPBA fail to show any selectivity, 1:1 mixtures of E‐ and Z‐olefins being formed.     

Environmentally friendly liquid phase oxidation: enhanced selectivity in the aerial oxidation of alkyl aromatics,epoxidations and the Baeyer–Villiger oxidation using novel silica supported transition metal ions

Active transition metal species (Co, Cu, Cr, Ni or Mn) supported on a chemically modified silica gel are used as heterogeneous catalysts in a range of liquid phase oxidation reactions: alkyl aromatic side chain oxidations, epoxidations of alkenes and Baeyer–Villiger oxidations of linear ketones to esters and cyclic ketones to lactones. The catalyst employs metal centres bound to the silica surface via a hydrophobic spacer chain and is thus chemically robust and has a relatively high loading for a supported reagent (c 0.4 mmol g⁻¹). The Cr version of the catalyst promotes the oxidation of ethylbenzene to acetophenone in a solvent‐free system at a rate of 5.5% h⁻¹ (>370 turnover h⁻¹). It is also active for the oxidation of p‐chlorotoluene and p‐xylene to p‐chlorobenzoic acid and p‐toluic acid respectively. Cyclohexene is converted to its oxide at room temperature at a rate of c 28% h⁻¹ (c 12 turnover h⁻¹) using either the Ni or Cu versions of the catalyst. The room temperature Baeyer–Villiger oxidation of cyclohexanone is achieved at a rate of 44% h⁻¹ (49 turnover h⁻¹) using the Ni‐containing catalyst. The same material also promotes the Baeyer–Villiger oxidation of linear aliphatic ketones and aromatic side chains. All the above systems use either air or molecular oxygen as the oxidant rather than peroxides or peracids. © 1999 Society of Chemical Industry     

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.     

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.     

Biosynthesis of Hygrocins,Antitumor Naphthoquinone Ansamycins Produced by Streptomyces sp. LZ35

Hygrocins are naphthoquinone ansamycins with significant antitumor activities. Here, we report the identification and characterization of the hygrocin biosynthetic gene cluster (hgc) in Streptomyces sp. LZ35. A biosynthetic pathway is proposed based on bioinformatics analysis of the hgc genes and intermediates accumulated in selected gene disruption mutants. One of the steps during the biosynthesis of hygrocins is a Baeyer–Villiger oxidation between C5 and C6, catalyzed by luciferase‐like monooxygenase homologue Hgc3. Hgc3 represents the founding member of a previously uncharacterized family of enzymes acting as Baeyer–Villiger monooxygenases.     

The Catalytic Baeyer–Villiger Oxidation of Cyclohexanone to ε-Caprolactone over Stibium-containing Hydrotalcite

A series of hydrotalcite-like compounds were prepared under microwave irradiation, which were used to catalyze the Baeyer–Villiger oxidation of cyclohexanone to ε-caprolactone with hydrogen peroxide as oxidant. The results show that stibium-containing hydrotalcite (Sb-HTL) has good catalytic properties in the reaction. In the Baeyer–Villiger oxidation of cyclohexanone to ε-caprolactone with H₂O₂ catalyzed by Sb-HTL, the effects of reaction time, reaction temperature, amount of catalyst and H₂O₂/cyclohexanone molar ratio are also investigated in details. It is shown the cyclohexanone conversion and ε-caprolactone selectivity can reach 79.15 and 93.84%, respectively, under the optimum reaction conditions. Furthermore, Sb-HTL can be reused for six times without obvious loss of activity and selectivity. Therefore, Sb-HTL is reusable and would be a promising catalyst for the Baeyer–Villiger oxidation using green and cheap oxidants like hydrogen peroxide instead of peroxycarboxylic acids.     

Catalysis by Dihydrofolate Reductase from the Psychropiezophile Moritella profunda

The influence of temperature and pH on the stability and catalytic activity of dihydrofolate reductase (MpDHFR) from the cold‐adapted deep‐sea bacterium Moritella profunda was studied. The thermal melting temperature was found to be ～38 °C and was not affected by pH, while activity measurements demonstrated that its stability was maximal at pH 7 and was reduced dramatically below pH 6 or above pH 8. The steady‐state rate constant (k_cat) was maximal at neutral pH and higher temperatures, while the Michaelis constants (K_M) for both substrate and cofactor were optimal at lower temperatures and at elevated or reduced pH. For both temperature and pH, any change in k_cat was therefore offset by a similar change in K_M. Both the activation enthalpy and entropy of the MpDHFR‐catalysed reaction were lower than those of DHFR from E. coli leading overall to a very small difference in activation free energy and therefore similar steady‐state rate constants at the same temperature. The chemical step of the reaction is not rate limiting at pH 7, but becomes progressively more rate limiting as the pH increases. These results demonstrate adaptation of MpDHFR to its environment and show compromises between enthalpic and entropic contributions to the reaction, and between k_cat and K_M.     

Exploring the Biocatalytic Scope of Alditol Oxidase from Streptomyces coelicolor

The substrate scope of the flavoprotein alditol oxidase (AldO) from Streptomyces coelicolor A3(2), recombinantly produced in Escherichia coli, was explored. While it has been established that AldO efficiently oxidizes alditols to D ‐aldoses, this study revealed that the enzyme is also active with a broad range of aliphatic and aromatic alcohols. Alcohols containing hydroxy groups at the C‐1 and C‐2 positions like 1,2,4‐butanetriol (K_m=170 mM, k_cat=4.4 s⁻¹), 1,2‐pentanediol (K_m=52 mM, k_cat=0.85 s⁻¹) and 1,2‐hexanediol (K_m=97 mM, k_cat=2.0 s⁻¹) were readily accepted by AldO. Furthermore, the enzyme was highly enantioselective for the oxidation of 1,2‐diols [e.g., for 1‐phenyl‐1,2‐ethanediol the (R)‐enantiomer was preferred with an E‐value of 74]. For several diols the oxidation products were determined by GC‐MS and NMR. Interestingly, for all tested 1,2‐diols the products were found to be the α‐hydroxy acids instead of the expected α‐hydroxy aldehydes. Incubation of (R)‐1‐phenyl‐1,2‐ethanediol with ¹⁸O‐labelled water (H₂¹⁸O) revealed that a second enzymatic oxidation step occurs via the hydrate product intermediate. The relaxed substrate specificity, excellent enantioselectivity, and independence of coenzymes make AldO an attractive enzyme for the preparation of optically pure 1,2‐diols and α‐hydroxy acids.     

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.     

Unexpected NADPH Hydratase Activity in the Nitrile Reductase QueF from Escherichia coli

The nitrile reductase QueF catalyzes NADPH-dependent reduction of the nitrile group of preQ₀ (7-cyano-7-deazaguanine) into the primary amine of preQ₁ (7-aminomethyl-7-deazaguanine), a biologically unique reaction important in bacterial nucleoside biosynthesis. Here we have discovered that the QueF from Escherichia coli—its D197A and E89L variants in particular (apparent k_cat≈10⁻² min⁻¹)—also catalyze the slow hydration of the C5=C6 double bond of the dihydronicotinamide moiety of NADPH. The enzymatically C6-hydrated NADPH is a 3.5:1 mixture of R and S forms and rearranges spontaneously through anomeric epimerization (β→α) and cyclization at the tetrahydronicotinamide C6 and the ribosyl O2. NADH and 1-methyl- or 1-benzyl-1,4-dihydronicotinamide are not substrates of the enzymatic hydration. Mutagenesis results support a QueF hydratase mechanism, in which Cys190—the essential catalytic nucleophile for nitrile reduction—acts as the general acid for protonation at the dihydronicotinamide C5 of NADPH. Thus, the NADPH hydration in the presence of QueF bears mechanistic resemblance to the C=C double bond hydration in natural hydratases.     

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.     

Application of a thermostable Baeyer–Villiger monooxygenase for the synthesis of branched polyester precursors

BACKGROUND

It is widely accepted that the poor thermostability of Baeyer–Villiger monooxygenases limits their use as biocatalysts for applied biocatalysis in industrial applications. The goal of this study was to investigate the biocatalytic oxidation of 3,3,5‐trimethylcyclohexanone using a thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) for the synthesis of branched ?‐caprolactone derivatives as building blocks for tuned polymeric backbones. In this multi‐enzymatic reaction, the thermostable cyclohexanone monooxygenase was fused to a phosphite dehydrogenase (PTDH) in order to ensure co‐factor regeneration.

RESULTS

Using reaction engineering, the reaction rate and product formation of the regio‐isomeric branched lactones were improved and the use of co‐solvents and the initial substrate load were investigated. Substrate inhibition and poor product solubility were overcome using continuous substrate feeding regimes, as well as a biphasic reaction system with toluene as water‐immiscible organic solvent. A maximum volumetric productivity, or space–time‐yield, of 1.20 g L^‐1 h^‐1 was achieved with continuous feeding of substrate using methanol as co‐solvent, while a maximum product concentration of 11.6 g L^‐1 was achieved with toluene acting as a second phase and substrate reservoir.

CONCLUSION

These improvements in key process metrics therefore demonstrate progress towards the up‐scaled Baeyer–Villiger monooxygenase‐biocatalyzed synthesis of the target building blocks for polymer application. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

     

Aureobasidium subglaciale F134 is a bifunctional whole-cell biocatalyst for Baeyer–Villiger oxidation or selective carbonyl reduction controllable by temperature

The microbial production of either ester/lactones or enantio-enriched alcohols through Baeyer–Villiger oxidation or stereoselective reduction of ketones, respectively, is possible by using whole cells of A. subglaciale F134 as a bifunctional biocatalyst. The chemoselective pattern of acetophenone biotransformation catalyzed by these cells can be regulated through reaction temperature, directing the reaction either towards oxidation or reduction products. The Baeyer–Villiger oxidation activity of A. subglaciale F134 whole cells is particularly dependent on reaction temperature. Acetophenone was transformed efficiently to phenol via the primary Baeyer–Villiger product phenyl acetate at 20 °C after 48 h with 100% conversion. In contrast, at 35 °C, enantio-enriched (S)-1-phenylethanol was obtained as the sole product with 64% conversion and 89% ee. In addition, A. subglaciale F134 cells also catalyze the selective reduction of various structurally different aldehydes and ketones to alcohols with 40% to 100% yield, indicating broad substrate spectrum and good enantioselectivity in relevant cases. Our study provides a bifunctional biocatalyst system that can be used in Baeyer–Villiger oxidation as well as in asymmetric carbonyl reduction, setting the stage for future work concerning the identification and isolation of the respective enzymes.     

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.     

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.     

Catalysis of an Essential Step in Vitamin B2 Biosynthesis by a Consortium of Broad Spectrum Hydrolases

An enzyme catalysing the essential dephosphorylation of the riboflavin precursor, 5‐amino‐6‐ribitylamino‐2,4(1H,3H)‐pyrimidinedione 5′‐phosphate ( 6 ), was purified about 800‐fold from a riboflavin‐producing Bacillus subtilis strain, and was assigned as the translation product of the ycsE gene by mass spectrometry. YcsE is a member of the large haloacid dehalogenase (HAD) superfamily. The recombinant protein was expressed in Escherichia coli. It catalyses the hydrolysis of 6 (v_max, 12 μmol mg^?1 min^?1; K_M, 54 μm ) and of FMN (v_max, 25 μmol mg^?1 min^?1; K_M, 135 μm ). A ycsE deletion mutant of B. subtilis was not riboflavin dependent. Two additional proteins (YwtE, YitU) that catalyse the hydrolysis of 6 at appreciable rates were identified by screening 13 putative HAD superfamily members from B. subtilis. The evolutionary processes that have resulted in the handling of an essential step in the biosynthesis of an essential cofactor by a consortium of promiscuous enzymes require further analysis.     

Unusual CC Bond Isomerization of an α,β‐Unsaturated γ‐Butyrolactone Catalysed by Flavoproteins from the Old Yellow Enzyme Family

An unexpected, redox‐neutral C?C bond isomerization of a γ‐butyrolactone bearing an exo‐methylene unit to the thermodynamically more favoured endo isomer (k_cat=0.076 s^?1) catalysed by flavoproteins from the Old Yellow Enzyme family was discovered. Theoretical calculations and kinetic data support a mechanism through which the isomerization proceeds through FMN‐mediated hydride addition onto exo‐Cβ, followed by hydride abstraction from endo‐Cβ′, which is in line with the well‐established C?C bond bioreduction of OYEs. This new isomerase activity enriches the catalytic versatility of ene‐reductases.     

Characterization of the Aminotransferase ThdN from Thienodolin Biosynthesis in Streptomyces albogriseolus

In Streptomyces albogriseolus the indolethiophen alkaloid thienodolin is derived from tryptophan. The first step in thienodolin biosynthesis is the regioselective chlorination of tryptophan in the 6‐position of the indole ring. The second step is catalyzed by the aminotransferase ThdN. ThdN shows sequence homology (up to 69 % similarity) with known pyridoxal 5′‐phosphate‐dependent aminotransferases of the aspartate aminotransferase family from Gram‐positive bacteria. thdN was heterologously expressed in Pseudomonas fluorescens, and the enzyme was purified by nickel‐affinity chromatography. ThdN is a homodimeric enzyme with a mass of 90 600 kDa and catalyzes the conversion of l ‐tryptophan and a number of chlorinated and brominated l ‐tryptophans. The lowest K_M values were found for 6‐bromo‐ and 6‐chlorotryptophan (40 and 66 μm , respectively). For l ‐tryptophan it was 454 μm, which explains why thienodolin is the major product and dechlorothienodolin is only a minor component. The turnover number (k_cat) for 7‐chlorotryptophan (128 min^?1) was higher than that for the natural substrate 6‐chlorotryptophan (88 min^?1).     

Cloning,Expression and Purification of Cindoxin,an Unusual Fmn‐Containing Cytochrome P450 Redox Partner

Cytochromes P450 (P450s) belong to a superfamily of haemoproteins that catalyse a remarkable variety of oxidative transformations. P450 catalysis generally requires that cognate redox proteins transfer electrons, derived ultimately from NAD(P)H, to the P450 for oxygen activation. P450_cin (CYP176A1) is a bacterial P450 that is postulated to allow Citrobacter braakii to live on cineole as its sole carbon source by initiating cineole biodegradation. Here we report the cloning, expression, purification and characterisation of one of its postulated redox partners, cindoxin (Cdx), which has strong similarity to the FMN domain of cytochrome P450 reductase. Cindoxin reductase (CdR), which displays strong similarity to NADPH‐dependent ferredoxin reductases, was unable to be expressed in a functional form. Mass spectrometric and HPLC analyses confirmed that the flavin cofactor of cindoxin was FMN. Redox potentiometric titrations were performed with cindoxin within the range 6<pH<8; this enabled the quinone/semiquinone (E₁) and semiquinone/hydroquinone (E₂) redox potentials to be determined. Our results show that cindoxin might be somewhat different to other flavodoxins that interact with P450s, in which generally only one couple is important. Both redox states of cindoxin could be catalytically relevant. A catalytically active system was reconstituted in vitro with E. coli flavodoxin reductase (Fpr) acting as the terminal redox partner in the absence of CdR. Our results show that Cdx and Fpr support regio‐ and stereoselective P450_cin‐catalysed cineole oxidation to (1R)‐6β‐hydroxycineole with turnover rates up to 1500 min^?1. This system is tightly coupled with 80 % of NADPH reducing equivalents funnelled into substrate oxidation.