Ling Hua

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.

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).

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.

How carbonyl reductases control stereoselectivity: Approaching the goal of rational design

Although Prelog’s rule and two hydrophobic binding pockets model have been used to predict and explain the stereoselectivity of enzymatic ketone reduction, the molecular basis of stereorecognition by carbonyl reductases has not been well understood. The stereoselectivity is not only determined by the structures of enzymes and substrates, but also affected by the reaction conditions such as temperature and reaction medium. Structural analysis coupled with site-directed mutagenesis of stereocomplementary carbonyl reductases readily reveals the key elements of controlling stereoselectivity in these enzymes. In our studies, enzyme-substrate docking and molecular modeling have been engaged to understand the enantioselectivity diversity of the carbonyl reductase from Sporobolomyces salmonicolor (SSCR), and to guide site-saturation mutagenesis for altering the enantioselectivity of this enzyme. These studies provide valuable information for our understanding of how the residues involved in substrate binding affect the orientation of bound substrate, and thus control the reaction stereoselectivity. The in silico docking-guided semi-rational approach should be a useful methodology for discovery of new carbonyl reductases.

Influence of Cofactor Regeneration Strategies on Preparative-Scale, Asymmetric Carbonyl Reductions by Engineered Escherichia coli.

This study was designed to determine whether whole cells or crude enzyme extracts are more effective for preparative-scale ketone reductions by dehydrogenases as well as learning which cofactor regeneration scheme is most effective. Based on results from three representative ketone substrates (an α-fluoro-β-keto ester, a bis-trifluoromethylated acetophenone, and a symmetrical β-diketone), our results demonstrate that several nicotinamide cofactor regeneration strategies can be applied to preparative-scale dehydrogenase-catalyzed reactions successfully.