Protein-engineered bio-catalysis has become an efficient approach for synthesis of environmental friendly fine chemicals and pharmaceutical intermediates. With revolutionary advances in genome sequencing, gene synthesis and computational technologies, it is not difficult to identify a potentially useful biocatalyst for specific chemical reactions. However, natural enzymes often can’t meet the industry requirements. Therefore, enzyme engineering regarding improvement of the stability, activity, substrate specificity and enantioselectivity to obtain an effective biocatalyst is of increasing importance. Considering the industrial requirements for stability at high temperatures and pressures, as well as high concentrations of chemical denaturants, the thermostable enzymes have great potential in bio-catalysis. Thus, we are especially interested in the structure-based engineering of those thermostable microbial enzymes. This direction is supported by funds from “Key Laboratory of New Drug Discovery” (2014-2017). 

NADP(H)-dependent aldo-keto reductase

A novel NADP(H)-dependent aldo-keto reductase Tm1743 was identified from Thermotoga maritime, characterized with high thermostability, strong chemical tolerance and broad substrate specificity. It effectively catalyzes the asymmetric reduction of ketones and aldehydes to chiral alcohols, one of the most important building blocks for many pharmaceutical intermediates. However, poor enantioselectivity towards few important substrates greatly limited its application. We determined the crystal structures of Tm1743 in complex with NADP⁺ at 2.0 Å resolution (Acta Crystallogr F. 2015) and with two different inhibitors beyond 1.6 Å resolutions (FEBS Lett, 2019). Based on the structures, we extensively investigated the enantioselectivity of Tm1743 through molecular dynamic simulations and semi-rational design of the enzyme. Finally, we identified the best (R)- and (S)-enantiomer preferred mutant enzymes to produce the optical pure intermediates for angiotensin drug synthesis, with their enantiomeric excess values of 99.4% and 99.6% respectively. Our study demonstrates an efficient strategy to improve the enantioselectivity of a natural biocatalyst, it is meaningful to the exploration of novel green catalyst for classic asymmetric reactions (Sci Rep, 2017).

PLP-dependent alanine racemase

Pyridoxal 5’-phosphate (PLP) dependent alanine racemase catalyzes racemization of L-Ala to D-Ala, a key component of the peptidoglycan network in bacterial cell wall. It has been extensively studied as an important antimicrobial drug target due to its restriction in eukaryotes. However, many marketed alanine racemase inhibitors also act on eukaryotic PLP-dependent enzymes and cause side effects. A thermostable alanine racemase (Alr_Tt) from Thermoanaerobacter tengcongensis MB4 contains an evolutionarily non-conserved residue Gln360 in inner layer of the substrate entryway, which is supposed to be a key determinant in substrate specificity. We determined the crystal structure of Alr_Tt in complex with L-Ala at 2.7 Å resolution, and investigated the role of Gln360 by saturation mutagenesis and kinetic analysis. Compared to typical bacterial alanine racemase, presence of Gln360 and conformational changes of active site residues disrupted the hydrogen bonding interactions necessary for proper PLP immobilization, and decreased both the substrate affinity and turnover number of Alr_Tt. However, it could be complemented by introduction of hydrophobic amino acids at Gln360, through steric blocking and interactions with a hydrophobic patch near active site pocket. These observations explained the low racemase activity of Alr_Tt, revealed the essential role of Gln360 in substrate selection, and its preference for hydrophobic amino acids especially Tyr in bacterial alanine racemization. Our work will contribute new insights into the alanine racemization mechanism for antimicrobial drug development (PLoS One. 2015).