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Applied and Environmental Microbiology, July 2003, p. 3710-3718, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.3710-3718.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Jung-Kul Lee,^1* Sang-Yong Kim,² Yeon-Woo Ryu,³ Jin-Ho Seo,⁴ and Jung-Hoe Kim⁵

BioNgene Co., Ltd., Myungryun-Dong, Jongro-Ku, Seoul 110-521,¹ Bolak Co., Ltd., Kyongki-Do 445-930,² Department of Molecular Science and Technology, Ajou University, Suwon 442-749,³ Department of Food Science and Technology, Research Center for New Biomaterials in Agriculture, Seoul National University, Suwon 441-744,⁴ Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yuseong-Ku, Daejeon 305-701, Korea⁵

Received 6 January 2003/ Accepted 15 April 2003

Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K_m = 7.9 mM; k_cat/K_m = 0.73 mM^-1 s^-1). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k_cat/K_m = 450 mM^-1 s^-1) than with NADPH (k_cat/K_m = 5.5 mM^-1 s^-1), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu²⁺ and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.

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* Corresponding author. Mailing address: BioNgene Co., Ltd., 10-1 1Ka Myungryun-Dong, Jongro-Ku, Seoul 110-521, Korea. Phone: 82-2-747-0700. Fax: 82-2-747-0750. E-mail: jkrhee{at}biongene.com.

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Applied and Environmental Microbiology, July 2003, p. 3710-3718, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.3710-3718.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.