P-keto ester reductase

In this chapter, we describe development of novel enzymes including engineered PAR, LSADH, and P-keto ester reductase (KER) from P. citrinum key advances in tailoring biocatalysts by protein engineering and future aspect of them for improved bioreduction of ketones to synthesize various chiral alcohols. 

Engineering of p-Keto Ester Reductase (KER) for Raising Thermal Stability and Stereoselectivity... 

Baker s yeast has been widely used for the reduction of ketones. The substrate specificity and enantioselectivity of the carbonyl reductase from baker s yeast, which is known to catalyze the reduction of P-keto ester to L-hydroxyester (L2-enzyme) [15], was investigated, and the enzyme was found to reduce chloro-, acetoxy ketones with high enantioselectivity (Figure 8.32) [24aj. 

Glucose-grown cells of G. candidum SC 5469 have also catalyzed the stereoselective reduction of ethyl-, isopropyl-, and tertiary-butyl esters of 4-chloro-3-oxobutanoic acid and methyl and ethyl esters of 4-bromo-3-oxobutanoic acid. A reaction yield of >85% and e.e. of >94% were obtained. NAD+-depen-dent oxido-reductase responsible for the stereoselective reduction of P-keto esters of 4-chloro- and 4-bromo-3-oxobutanoic acid was purified 100-fold. The molecular weight of purified enzyme is 950,000. The purified oxido-reductase was immobilized on Eupergit C and used to catalyze the reduction of (39) to S-( - )-(40). The cofactor NAD+ required for the reduction reaction was regenerated by glucose dehydrogenase. 

Itoh, N., Asako, H., Barmo, K., Makino, Y, Wakita, R., and Shimizu, M. (2004) Purification and characterization of NADPH-dependent aldo-keto reductase specific for P-keto esters from PenuMium citrirmm. Appl. Microbiol. Biotechnol., 66, 53-62. 

Xu and coworkers reported several robust carbonyl reductases discovered via in silico data rnining approaches and developed a set of water-based bioreduction processes for efficient synthesis of functionalized optically active a- and P-hydroxy esters [2-4]. As shown in Table 9.1, several a- and P-keto esters were asymmetrically reduced at high loads using a carbonyl reductase mined from Candida abrata... 

A few years later, the same research group demonstrated the bioreduction of a-chloro-P keto esters 29-33. The production of at least two of the four possible a-chlorO P-hydroxy ester diastereomers with high stereoselectivities, by using whole cells of an E. coli strain overexpressing a single yeast reductase identified from screening studies, was successfully accomplished (Scheme 12.16) [33]. 

So far, most microorganisms and enzymes derived therefrom have been used in the reduction of a single keto group of p-keto or a-keto compounds [68-71], Recently, Patel et al. [72] have demonstrated the stereoselective reduction of 3,5-dioxo-6-(benzyloxy)hexanoic acid, ethyl ester (41), to (3,S, 5R)-dihydroxy-6-(benzyloxy)hexanoic acid, ethyl ester (42a) (Fig. 14). The compound (42a) is a key chiral intermediate required for the chemical synthesis of [4-[4a,6P(E)]]-6-[4,4-bis(4-fluorophenyl)-3-(l-methyl-lH-tetrazol-5-yl)-l,3-butadienyl]-tetrahydro-4-hydroxy-2H-pyran-2-one, compound R-( + )-(43), a new anticholesterol drug that acts by inhibition of HMG CoA reductase [73], Among various microbial cultures evaluated for the stereoselective reduction of diketone (41), cell suspensions of Aci-... 

The enzyme giving (ft)-4-chIoro-3-hydroxybutanoate esters was isolated in crystalline form from S. salmonicolor and was characterized in some detail [111-113], It is a kind of aldehyde reductase with a molecular mass of about 37,000. The enzyme absolutely requires NADPH as a cofactor. Besides 4-haloacetoacetate esters, p-nitrobenzaldehyde and a variety of other aromatic and aliphatic aldehydes have been found to be reduced well by the enzyme (see Tables 4 and 5). The structural gene of the enzyme was cloned and the enzyme has shown to have similarity to the mammalian aldo-keto-reductase superfamily enzymes in primary protein structure [114], In S. salmonicolor, the enzyme comprised 2 6% of the total extractable proteins. Such a high content of the enzyme suggests that this may be one of the reasons why the yeast catalyzes conversion to the (R)-enantiomer predominantly. 

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