Structural insights into substrate and coenzyme preference by SDR family protein Gox2253 from Gluconobater oxydans.
Gox2253 from Gluconobacter oxydans belongs to the short-chain dehydrogenases/reductases family, and catalyzes the reduction of heptanal, octanal, nonanal, and decanal with NADPH. To develop a robust working platform to engineer novel G. oxydans oxidoreductases with designed coenzyme preference, we adopted a structure based rational design strategy using computational predictions that considers the number of hydrogen bonds formed between enzyme and docked coenzyme. We report the crystal structure of Gox2253 at 2.6 Å resolution, ternary models of Gox2253 mutants in complex with NADH/short-chain aldehydes, and propose a structural mechanism of substrate selection. Molecular dynamics simulation shows that hydrogen bonds could form between 2'-hydroxyl group in the adenosine moiety of NADH and the side chain of Gox2253 mutant after arginine at position 42 is replaced with tyrosine or lysine. Consistent with the molecular dynamics prediction, Gox2253-R42Y/K mutants can use both NADH and NADPH as a coenzyme. Hence, the strategies here could provide a practical platform to engineer coenzyme selectivity for any given oxidoreductase and could serve as an additional consideration to engineer substrate-binding pockets.
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China; Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, Singapore, 117543, Singapore.
Bo
B
Yin
Dongbing
D
Cui
Lujia
L
Zhang
Shuiqin
S
Jiang
Satoru
S
Machida
Y Adam
YA
Yuan
Dongzhi
D
Wei
D000447:Aldehydes / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003067:Coenzymes / Q000378:metabolism; D018360:Crystallography, X-Ray; D020600:Gluconobacter oxydans / Q000737:chemistry*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000447:Aldehydes; D001426:Bacterial Proteins; D003067:Coenzymes; D009249:NADP; C479548:undecanal; D010088:Oxidoreductases
G. oxydans Gox2253; coenzyme preference; crystal structure; short-chain dehydrogenases/reductases; substrate selection