Qiaqing Wu

A Novel meso-Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum: Overexpression, Characterization, and Potential for d-Amino Acid Synthesis

ABSTRACT meso-Diaminopimelate dehydrogenase (meso-DAPDH) is an NADP+-dependent enzyme which catalyzes the reversible oxidative deamination on the d-configuration of meso-2,6-diaminopimelate to produce l-2-amino-6-oxopimelate. In this study, the gene encoding a meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum was cloned and expressed in Escherichia coli. In addition to the native substrate meso-2,6-diaminopimelate, the purified enzyme also showed activity toward d-alanine, d-valine, and d-lysine. This enzyme catalyzed the reductive amination of 2-keto acids such as pyruvic acid to generate d-amino acids in up to 99% conversion and 99% enantiomeric excess. Since meso-diaminopimelate dehydrogenases are known to be specific to meso-2,6-diaminopimelate, this is a unique wild-type meso-diaminopimelate dehydrogenase with a more relaxed substrate specificity and potential for d-amino acid synthesis. The enzyme is the most stable meso-diaminopimelate dehydrogenase reported to now. Two amino acid residues (F146 and M152) in the substrate binding sites of S. thermophilum meso-DAPDH different from the sequences of other known meso-DAPDHs were replaced with the conserved amino acids in other meso-DAPDHs, and assay of wild-type and mutant enzyme activities revealed that F146 and M152 are not critical in determining the enzymes substrate specificity. The high thermostability and relaxed substrate profile of S. thermophilum meso-DAPDH warrant it as an excellent starting enzyme for creating effective d-amino acid dehydrogenases by protein engineering.

Biochemical characterization and substrate profiling of a new NADH-dependent enoate reductase from Lactobacillus casei

Carbon-carbon double bond of α,β-unsaturated carbonyl compounds can be reduced by enoate reductase (ER), which is an important reaction in fine chemical synthesis. A putative enoate reductase gene from Lactobacillus casei str. Zhang was cloned into pET-21a+ and expressed in Escherichia coli BL21 (DE3) host cells. The encoded enzyme (LacER) was purified by ammonium sulfate precipitation and treatment in an acidic buffer. This enzyme was identified as a NADH-dependent enoate reductase, which had a K(m) of 0.034 ± 0.006 mM and k(cat) of (3.2 ± 0.2) × 10³ s⁻¹ toward NADH using 2-cyclohexen-1-one as the substrate. Its K(m) and k(cat) toward substrate 2-cyclohexen-1-one were 1.94 ± 0.04 mM and (8.4 ± 0.2) × 10³ s⁻¹, respectively. The enzyme showed a maximum activity at pH 8.0-9.0. The optimum temperature of the enzyme was 50-55°C, and LacER was relatively stable below 60 °C. The enzyme was active toward aliphatic alkenyl aldehyde, ketones and some cyclic anhydrides. Substituted groups of cyclic α,β-unsaturated ketones and its ring size have positive or negative effects on activity. (R)-(-)-Carvone was reduced to (2R,5R)-dihydrocarvone with 99% conversion and 98% (diasteromeric excess: de) stereoselectivity, indicating a high synthetic potential of LacER in asymmetric synthesis.

Characterization of (R)-selective amine transaminases identified by in silico motif sequence blast

Compared to (S)-selective amine transaminase ((S)-AT), the (R)-selective counterpart ((R)-AT) has been less studied. As such, a simplified “Motif Sequence Blast” search (Höhne et al. Nat Chem Biol 6:807–813, 2010) was carried out to identify new (R)-ATs from the protein databases. The combined conserved sequence motifs of (R)-ATs based on the previous in silico method of predicting (R)-selective amine transaminase were used as the template sequence for BLASTP search at default settings in NCBI, and six candidate sequences were identified. These putative (R)-AT genes were synthesized and overexpressed in Escherichia coli. Among them, five new (R)-ATs were expressed as soluble protein and showed unusual substrate specificity and high stereoselectivity. Furthermore, several unnatural amino acids, such as d-alanine, d-2-aminobutyric acid, and d-norvaline, were synthesized via the (R)-AT-catalyzed amino transfer reaction to the corresponding keto acids. Optically pure (S)-amines were also obtained by kinetic resolution of racemic amines catalyzed with these new (R)-ATs. Therefore, the Motif Sequence Blast search offers a quick and effective method for in silico identification of new (R)-ATs, and the newly identified (R)-ATs are attractive additions to the toolbox of (R)-ATs for further study and industrial application.

Engineering the meso-Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum by Site Saturation Mutagenesis for d-Phenylalanine Synthesis

ABSTRACT In order to enlarge the substrate binding pocket of the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum to accommodate larger 2-keto acids, four amino acid residues (Phe146, Thr171, Arg181, and His227) were targeted for site saturation mutagenesis. Among all mutants, the single mutant H227V had a specific activity of 2.39 ± 0.06 U · mg−1, which was 35.1-fold enhancement over the wild-type enzyme.

Structural and Mutational Studies on the Unusual Substrate Specificity of meso‐Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum

Wild‐type meso‐diaminopimelate dehydrogenase (DAPDH) is usually specific to the native substrate, meso‐2,6‐diaminopimelate. Recently, a DAPDH from Symbiobacterium thermophilum (StDAPDH) was found to exhibit expanded substrate specificity. As such, its crystal structures in apo form and in complex with NADP+ and both NADPH and meso‐DAP were investigated to reveal the structural basis of its unique catalytic properties. Structural analysis results show that StDAPDH should prefer an ordered kinetic catalytic mechanism. A second substrate entrance tunnel with Met152 at its bottleneck was found, through which pyruvate/D‐alanine might bind and enter the catalytic cavity, providing some structural insights into its high activity toward pyruvate. The side chain of Met152 might interact with Asp92 and Asn253, thus affecting the domain motion and catalysis. These results offer useful information for understanding the unique catalytic properties of StDAPDH and guiding further engineering of this enzyme.

Novel preparation protocol for the expression and purification of recombinant staphylokinase.

Staphylokinase (Sak), produced by lysogenic strains of Staphylococcus aureus, can convert plasminogen into its proteolytic form, plasmin, and thus is widely used to dissolve pathological clots in clinical applications. In the present paper, we report a novel approach to produce r‐Sak (recombinant Sak) using an engineered Escherichia coli expression system. The expression plasmid was constructed by placing the Sak gene into the expression vector pET32(a), resulting in the expression of 35% fusion protein. Subsequently, a rapid and simple chromatographic procedure was developed for the large‐scale purification of therapeutic‐grade r‐Sak from E. coli, which includes Ni2+‐affinity chromatography, ultrafiltration and Q‐Sepharose Fast Flow chroma‐tography. This method led to the production of highly pure r‐Sak (>99%), according to SDS/PAGE and HPLC analysis.

The catalytic promiscuity of a microbial 7α-hydroxysteroid dehydrogenase. Reduction of non-steroidal carbonyl compounds

A thermostable 7α-hydroxysteroid dehydrogenase from Bacteroides fragilis ATCC 25285 was found to catalyze the reduction of various benzaldehyde analogues to their corresponding benzyl alcohols. The enzyme activity was dependent upon the substituent on the benzene ring of the substrates. Benzaldehydes with electron-withdrawing substituent usually showed higher activity than those with electron-donating groups. Furthermore, this enzyme was tolerant to some organic solvents. These results together with previous studies suggested that 7α-hydroxysteroid dehydrogenase from B. fragilis might play multiple functional roles in biosynthesis and metabolism of bile acids, and in the detoxification of xenobiotics containing carbonyl groups in the large intestine. In addition, its broad substrate spectrum offers great potential for finding applications not only in the synthesis of steroidal compounds of pharmaceutical importance, but also for the production of other high-value fine chemicals.

Efficient Biosynthesis of Ethyl (R)‐3‐Hydroxyglutarate through a One‐Pot Bienzymatic Cascade of Halohydrin Dehalogenase and Nitrilase

An effective one‐pot bienzymatic synthesis of ethyl (R)‐3‐hydroxyglutarate (EHG) from ethyl (S)‐4‐chloro‐3‐hydroxybutyrate (ECHB) was achieved by using recombinant Escherichia coli cells expressing separately or co‐expressing a mutant halohydrin dehalogenase gene from Agrobacterium radiobacter AD1 and a nitrilase gene from Arabidopsis thaliana. The activity of nitrilase was inhibited by high concentration of ECHB and NaCN. Consequently, the one‐pot one‐step process was implemented by fed‐batch of ECHB and NaCN with high accumulative product concentration (up to 0.9 mol L−1). The biotransformation of ECHB to EHG was successfully achieved at 1.2 mol L−1 substrate concentration by a one‐pot two‐step process. As such, this one‐pot bienzymatic transformation should be useful in synthesizing these important optical pure β‐hydroxycarboxylic acids.

Synthesis of α,β-unsaturated esters via a chemo-enzymatic chain elongation approach by combining carboxylic acid reduction and Wittig reaction.

Summary α,β-Unsaturated esters are versatile building blocks for organic synthesis and of significant importance for industrial applications. A great variety of synthetic methods have been developed, and quite a number of them use aldehydes as precursors. Herein we report a chemo-enzymatic chain elongation approach to access α,β-unsaturated esters by combining an enzymatic carboxylic acid reduction and Wittig reaction. Recently, we have found that Mycobacterium sp. was able to reduce phenylacetic acid (1a) to 2-phenyl-1-ethanol (1c) and two sequences in the Mycobacterium sp. genome had high identity with the carboxylic acid reductase (CAR) gene from Nocardia iowensis. These two putative CAR genes were cloned, overexpressed in E. coli and one of two proteins could reduce 1a. The recombinant CAR was purified and characterized. The enzyme exhibited high activity toward a variety of aromatic and aliphatic carboxylic acids, including ibuprofen. The Mycobacterium CAR catalyzed carboxylic acid reduction to give aldehydes, followed by a Wittig reaction to afford the products α,β-unsaturated esters with extension of two carbon atoms, demonstrating a new chemo-enzymatic method for the synthesis of these important compounds.

Enzymatic Synthesis of a Key Intermediate for Rosuvastatin by Nitrilase-Catalyzed Hydrolysis of Ethyl (R)-4-Cyano-3-hydroxybutyate at High Substrate Concentration

An enzymatic method for the synthesis of ethyl (R)‐3‐hydroxyglutarate from ethyl (R)‐4‐cyano‐3‐hydroxybutyate was developed by using free and immobilized recombinant Escherichia coli BL21(DE3)pLysS harboring a nitrilase gene from Arabidopsis thaliana (AtNIT2). The hydrolysis of ethyl (R)‐4‐cyano‐3‐hydroxybutyate proceeded with the freely suspended cells of the biocatalyst under the optimized conditions of 1.5 mol L−1 (235.5 g L−1) substrate concentration and 6.0 wt % loading of wet cells at pH 8.0 and 25 °C, with 100 % conversion obtained in 4.5 h. Furthermore, immobilization of the whole cells enhanced their substrate tolerance, stability, and reusability. Under the optimized conditions (100 mmol L−1 tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 8.0, 25 °C), the immobilized biocatalyst could be reused for up to 16 batches, with a biocatalyst productivity of 55.6 g gwet cells−1 and a space‐time productivity of 625.5 g L−1 d−1. These results demonstrated that the immobilized whole cells might be used as a biocatalyst in the industrial production of ethyl (R)‐3‐hydroxyglutarate, a key intermediate for the synthesis of rosuvastatin.