(71b) Structure-Based Approach of Harnessing Aldo-Keto Reductases for Biocatalysis

Enzyme-catalyzed enantioselective reductions of ketones have become quite popular in the industrial organic chemistry for the production of homochiral alcohol building blocks. There is thus considerable interest in the development of novel biocatalysts with improved properties and expanded applications. If the already known substrate-activity patterns for different reductases could be correlated more clearly and systematically with their structure and function, the process of enzyme selection and design for novel biocatalytic reductions would be greatly facilitated and could therefore be accelerated considerably. This work aims at bridging the gap between fundamental studies of an oxidoreductase, the xylose reductase from Candida tenuis (CtXR), an aldo-keto reductase (AKR), and application of this enzyme in biocatalysis. The investigated enzyme and AKRs in general are due to their unusual broad substrate acceptance perfectly suited as biocatalysts. In this work a structure-based systematic examination of CtXR with respect to substrate specificity is presented.

How the AKRs bind and achieve stereoselectivity has generally remained vague. Modelling of xylose into the active site of CtXR suggested that Trp24, Asp51, and Asn310 are the main components of pentose-specific substrate binding recognition [1]. The molecular analysis of the substrate binding pocket of CtXR reveals that Phe115 and Met228, although not directly interacting with bound xylose, are also contributed to the site. Kinetic consequences of site-directed substitutions of these residues are reported. All mutants catalysed NADH-dependent reduction of xylose with reduced efficiency (k_cat/K_m) compared to the wild-type but improved the wild-type selectivity for utilisation of ketones, relative to xylose, by factors of 2 to 355-fold. The Trp24Phe mutant was further investigated for the production of industrially relevant alcohols in both cell-free systems [3] and whole cell reductions utilizing the hosts Saccharomyces cerevisiae [4] and Escherichia coli [5].

Enzyme, strain and reaction engineering was integrated to improve optical purity, efficiency and co-substrate yield in the reduction of &alpha-keto esters. The corresponding &alpha-hydroxy esters were obtained in good yields and excellent optical purities. Results from the two most popular hosts in whole cell reductions, Saccharomyces cerevisiae and Escherichia coli, were benchmarked against each other and pros and cons are extracted.

[1] Kavanagh, K., Klimacek, M., Nidetzky, B., Wilson, D.K. (2002) Biochemistry 41, 8785-8795.

[2] Kratzer, R., Leitgeb, S., Wilson, D.K., Nidetzky, B. (2006) Biochem. J. 393, 51-58.

[3] Kratzer, R., Nidetzky, B. (2007) Chem. Comm. 10, 1047-1049.

[4] Kratzer, R., Egger, S., Nidetzky, B. (2008) Biotechnol. Bioeng. 101, 1094-1101.

[5] Kratzer, R., Egger, S., Pukl, M., Nidetzky, B. (2008) Microb. Cell Fact. 10; 37-49.