Structural and mutational studies on an aldo-keto reductase AKR5C3 from Gluconobacter oxydans

Authors

  • Xu Liu,

    1. State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, China
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    • Xu Liu and Chao Wang contributed equally to this work

  • Chao Wang,

    1. Department of Biological Sciences and Center for Bioimaging Sciences, National University of Singapore, Singapore, Singapore
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    • Xu Liu and Chao Wang contributed equally to this work

  • Lujia Zhang,

    1. State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, China
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  • Zhiqiang Yao,

    1. State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, China
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  • Dongbing Cui,

    1. State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, China
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  • Liang Wu,

    1. State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, China
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  • Jinping Lin,

    Corresponding author
    1. State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, China
    • Correspondence to: Jinping Lin, 311# P.O. Box, 130 Meilong Road, Shanghai 200237, People's Republic of China. E-mail: jplin@ecust.edu.cn or Yu-Ren Adam Yuan, 14 Science Drive 4, Singapore 117543, Singapore. E-mail: dbsyya@nus.edu.sg

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  • Yu-Ren Adam Yuan,

    Corresponding author
    1. Department of Biological Sciences and Center for Bioimaging Sciences, National University of Singapore, Singapore, Singapore
    2. National University of Singapore (Suzhou) Research Institute, Suzhou Industrial Park, Jiangsu, China
    • Correspondence to: Jinping Lin, 311# P.O. Box, 130 Meilong Road, Shanghai 200237, People's Republic of China. E-mail: jplin@ecust.edu.cn or Yu-Ren Adam Yuan, 14 Science Drive 4, Singapore 117543, Singapore. E-mail: dbsyya@nus.edu.sg

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  • Dongzhi Wei

    1. State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, China
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  • The copyright line for this article was changed on 1 September 2014 after original online publication.

Abstract

An aldo-keto reductase AKR5C3 from Gluconobacter oxydans (designated as Gox0644) is a useful enzyme with various substrates, including aldehydes, diacetyl, keto esters, and α-ketocarbonyl compounds. The crystal structures of AKR5C3 in apoform in complex with NADPH and the D53A mutant (AKR5C3-D53A) in complex with NADPH are presented herein. Structure comparison and site-directed mutagenesis combined with biochemical kinetics analysis reveal that the conserved Asp53 in the AKR5C3 catalytic tetrad has a crucial role in securing active pocket conformation. The gain-of-function Asp53 to Ala mutation triggers conformational changes on the Trp30 and Trp191 side chains, improving NADPH affinity to AKR5C3, which helps increase catalytic efficiency. The highly conserved Trp30 and Trp191 residues interact with the nicotinamide moiety of NADPH and help form the NADPH-binding pocket. The AKR5C3-W30A and AKR5C3-W191Y mutants show decreased activities, confirming that both residues facilitate catalysis. Residue Trp191 is in the loop structure, and the AKR5C3-W191Y mutant does not react with benzaldehyde, which might also determine substrate recognition. Arg192, which is involved in the substrate binding, is another important residue. The introduction of R192G increases substrate-binding affinity by improving hydrophobicity in the substrate-binding pocket. These results not only supplement the AKRs superfamily with crystal structures but also provide useful information for understanding the catalytic properties of AKR5C3 and guiding further engineering of this enzyme.

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