Dunming Zhu

Inverting the Enantioselectivity of a Carbonyl Reductase via Substrate−Enzyme Docking-Guided Point Mutation

Substrate-enzyme docking-guided point mutation of a carbonyl reductase from Sporobolomyces salmonicolor led to mutant enzymes, which reversed the enantiopreference and enhanced the enantioselectivity toward the reduction of para-substituted acetophenones. Such a dramatic change in the enantioselectivity indicates that the 245 residue in the catalytic site plays a critical role in determining the enantioselectivity of these ketone reductions, providing valuable insight into our understanding of how residues involved in substrate binding affect the orientation of bound substrate and thus control the reduction stereoselectivity.

Synthesis of Optically Pure 2-Azido-1-arylethanols with Isolated Enzymes and Conversion to Triazole-Containing β-Blocker Analogues Employing Click Chemistry

Both antipodes of 2-azido-1-arylethanols were synthesized with excellent optical purity via enzymatic reduction of the corresponding alpha-azidoacetophenone derivatives catalyzed by a recombinant carbonyl reductase from Candida magnoliae ( CMCR) or an alcohol dehydrogenase from Saccharomyces cerevisiae ( Ymr226c). This provides an effective route to this class of important compounds in optically pure form. ( S)-2-Azido-1-( p-chlorophenyl)ethanols reacted with alkynes employing click chemistry to afford high yields of optically pure triazole-containing beta-adrenergic receptor blocker analogues with potential biological activity.

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.

Highly enantioselective mutant carbonyl reductases created via structure-based site-saturation mutagenesis.

A carbonyl reductase from Sporobolomyces salmonicolor reduced para-substituted acetophenones with low enantioselectivity. Enzyme-substrate docking studies revealed that residues M242 and Q245 were in close proximity to the para-substituent of acetophenones in the substrate binding site. Site-saturation mutagenesis of M242 or Q245, and double mutation of M242 and Q245 were performed in order to enhance the enzymes enantioselectivity toward the reduction of para-substituted acetophenones. Three Q245 mutants were obtained, which inverted the enantiopreference of product alcohols from (R)- to (S)-configuration with high ee values (Org. Lett. 2008, 10, 525-528). Four M242 mutant enzymes also showed greater preference for the formation of (S)-enantiomeric alcohols than the wild-type enzyme, but to a much less extent than Q245 mutants. M242/Q245 double variations not only greatly affect the enantiomeric purity of the product alcohols, but also invert the enantiopreference, demonstrating that these residues play a critical role in determining the enantioselectivity of these ketone reductions. The kinetic parameters of these mutant enzymes indicated that residues 242 and 245 also exert an effect on the catalytic activity of this carbonyl reductase. Highly enantioselective mutant carbonyl reductases were created by site-saturation mutagenesis, among which the one bearing double mutations, M242L/Q245P, showed the highest enantioselectivity that catalyzed the reduction of the tested para-substituted acetophenones to give (S)-enantiomeric products in ≥99% ee with only one exception of p-fluoroacetophenone (92% ee).

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.

Cloning, Protein Sequence Clarification, and Substrate Specificity of a Leucine Dehydrogenase from Bacillus sphaericus ATCC4525

Although an X-ray model sequence of a leucine dehydrogenase from Bacillus sphaericus ATCC4525 was reported, the amino acid sequence of this enzyme has not been confirmed. In the current study, this leucine dehydrogenase gene was cloned, sequenced, and over-expressed in Escherichia coli, and the protein sequence has been clarified. This leucine dehydrogenase is not identical with that of B. sphaericus IFO3525 because there are 16 different amino acid residues between these two proteins. Since the information on the catalytic properties of leucine dehydrogenase from B. sphaericus ATCC4525 has been limited, the recombinant enzyme was purified as His-tagged protein and further studied. This enzyme showed activity toward aliphatic substrates for both oxidative deamination and reductive amination and is an effective catalyst for the asymmetric synthesis of α-amino acids from the corresponding α-ketoacids.

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.