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Abstract: Chemoenzymatic deracemization was applied to prepare d‐valine from racemic valine ethyl ester or l‐valine ethyl ester in high yield (up to 95 %) with excellent optical purity (>99 % ee) by employing a newly evolved cyclohexylamine oxidase (CHAO) variant Y321I/M226T exhibiting catalytic efficiency that was 30 times higher than that of the wildtype CHAO. Interestingly, CHAO and its variants showed opposite enantioselectivity for valine ethyl ester and phenylalanine ethyl ester. [ABSTRACT FROM AUTHOR]

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily. [ABSTRACT FROM AUTHOR]

Canolol (4-vinylsyringol, VS), a potent antioxidant and an alkylperoxyl radical scavenger originally discovered in crude canola oil (rapeseed), is produced by decarboxylation of sinapic acid (SA) during canola seed roasting. Chemical syntheses of VS from SA require thermal or microwave induced decarboxylation in the presence of a base. A laboratory-evolved enzyme, designated SA decarboxylase (SAD), was developed in this study. In a biphasic bioreactor system, SAD was shown to produce VS from SA extracts prepared from canola meal with an overall yield of 3.0 mg VS per g of canola meal. In addition, we investigated the application of VS in polymerization to produce polyvinylsyringol (PVS) as a potential biodegradable polymer. The characteristics of PVS determined by thermogravimetric analysis, differential scanning calorimetry and nanoindentation tests are described. [ABSTRACT FROM AUTHOR]

Cyclohexylamine oxidase (CHAO) is a flavoprotein first described in Brevibacterium oxydans strain IH-35A that carries out the initial step of the degradation of the industrial chemical cyclohexylamine to cyclohexanone. We have cloned and expressed in Escherichia coli the CHAO-encoding gene (chaA) from B. oxydans, purified CHAO and determined the structures of both the holoenzyme form of the enzyme and a product complex with cyclohexanone. CHAO is a 50 kDa monomer with a PHBH fold topology. It belongs to the flavin monooxygenase family of enzymes and exhibits high substrate specificity for alicyclic amines and sec-alkylamines. The overall structure is similar to that of other members of the flavin monooxygenase family, but lacks either of the C- or N-terminal extensions observed in these enzymes. Active site features of the flavin monooxygenase family are conserved in CHAO, including the characteristic aromatic cage. Differences in the orientations of residues of the CHAO aromatic cage result in a substrate-binding site that is more open than those of its structural relatives. Since CHAO has a buried hydrophobic active site with no obvious route for substrates and products, a random acceleration molecular dynamics simulation has been used to identify a potential egress route. The path identified includes an intermediate cavity and requires transient conformation changes in a shielding loop and a residue at the border of the substrate-binding cavity. These results provide a foundation for further studies with CHAO aimed at identifying features determining substrate specificity and for developing the biocatalytic potential of this enzyme. [ABSTRACT FROM AUTHOR]

The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavouring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a β-barrel structure and two α-helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the β-barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site. [ABSTRACT FROM AUTHOR]

A putative α/β hydrolase fold-encoding gene (locus tag TTE1809) from the genome of Thermoanaerobacter tengcongensis was cloned and expressed in Escherichia coli as a possible source of thermostable feruloyl esterase (FAE) for the production of antioxidant phenolic acids from biomass. Designated as TtFAE, the 33-kDa protein was purified to apparent homogeneity. The lipase-like sequence characteristics of TtFAE and its substrate specificity towards methyl ferulate, methyl sinapate, and methyl p-coumarate classify it as a new member of the type A FAEs. At 75°C, the enzyme retained at least 95% of its original activity for over 80 min; at 80°C, its half-life was found to be 50 min, rendering TtFAE a highly thermostable protein. Under different hydrolytic conditions, ferulic acid (FA) was shown to be released from feruloylated oligosaccharides prepared from triticale bran. An estimated recovery of 68 mg FA/100 g triticale bran was demonstrated by a 30% release of the total FA from triticale bran within a 5-h incubation period. Both the oxygen radical absorbing capacity values of the feruloylated oligosaccharides and free FA were also determined. Overall, this work introduces a new bacterial member to the growing family of plant cell wall degrading FAEs that at present is largely of fungal origin, and it benchmarks the bioproduction of FA from triticale bran. [ABSTRACT FROM AUTHOR]

There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of ( R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3–5°C increase in the apparent melting temperature ( Tm) of the mutants over the native BcEST that has a Tm of 50°C was outperformed by TtEST, a naturally occurring homologue with a Tm of 65°C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the α/β-fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis. [ABSTRACT FROM AUTHOR]

Mycobacterium sp. strain THO100 was isolated from a morpholine-containing culture of activated sewage sludge. This strain was able to utilize pyrrolidine, morpholine, piperidine, piperazine, and 1,2,3,6-tetrahydropyridine as the sole sources of carbon, nitrogen, and energy. The degradation pathway of pyrrolidine as the best substrate for cellular growth was proposed based on the assays of substrate-induced cytochrome P450 and constitutive enzyme activities toward 4-aminobutyric acid (GABA) and succinic semialdehyde (SSA). Its 16S ribosomal RNA gene sequence (16S rDNA) was identical to that of Mycobacterium tokaiense ATCC 27282T. The morABC genes responsible for alicyclic amine degradation were nearly identical among different species of Mycobacteria. Remarkably, repetitive sequences at the intergenic spacer (IGS) region between morC and orf1’ were detected by comparison of the nearly identical mor gene cluster regions. Considering the strain activity for alicyclic amine degradation, the deleted 65-bp DNA segment did not significantly alter the open reading frames, and the expression and functions of the P450mor system remained unaltered. In addition, we found a spontaneous deletion of P450mor from another strain HE5 containing the archetypal mor gene cluster, which indicated a possible occurrence of DNA recombination to rearrange the DNA. [ABSTRACT FROM AUTHOR]

p-Cymene monooxygenase (CMO) from Pseudomonas putida F1 consists of a hydroxylase (CymA1) and a reductase component (CymA2) which initiate p-cymene (p-isopropyltoluene) catabolism by oxidation of the methyl group to p-isopropylbenzyl alcohol (p-cumic alcohol). To study the possible diverse range of substrates catalyzed by CMO, the cymA1A2 genes were cloned in an Escherichia coli pT7-5 expression system and the cells were used in transformation experiments. The tested substrates include different substituents on the aromatic ring at the 2 (ortho), 3 (meta) or 4 (para) position relative to the methyl moiety. As a result, a distinct preference was observed for substrates containing at least an alkyl or heteroatom substituent at the para-position of toluene. The conversion rate of 4-chlorotoluene or 4-methylthiotoluene to the corresponding benzyl alcohol was found to be as good as the canonical substrate, p-cymene. But 3-chlorotoluene, 4-fluorotoluene and 4-nitrotoluene were relatively poor substrates. CMO is also capable of producing styrene oxide from styrene. However, the oxidation of 4-chlorostyrene to 4-chlorostyrene oxide was by far the fastest among the substrates used in this study. The various biotransformation products were identified by a combined solid phase microextraction/gas chromatographic-mass spectrometric analytical technique. [ABSTRACT FROM AUTHOR]

Describes a novel two-component todST systems which activates expression of the toluene degradation pathway in Pseudomonas putida. Localization and inactivation of todST genes; Overexpression of TodT and DNA binding; Peptide dimerization.

A statement is amended in the article by Isupov et al. [(2015 ). Acta Cryst. D71, 2344–2353]. [ABSTRACT FROM AUTHOR]