title: Structural insights into stereospecific reduction of α, β-unsaturated carbonyl substrates by old yellow enzyme from Gluconobacter oxydans.
abstract: We report the crystal structure of old yellow enzyme (OYE) family protein Gox0502 (a.a 1-315) in free form at 3.3 Å. Detailed structural analysis revealed the key residues involved in stereospecific determination of Gox0502, such as Trp66 and Trp100. Structure-based computational analysis suggested the bulky side chains of these tryptophan residues may play important roles in product stereoselectivity. The introduction of Ile or Phe or Tyr mutation significantly reduced the product diastereoselectivity. We hypothesized that less bulky side chains at these critical residues could create additional free space to accommodate intermediates with different conformations. Notably, the introduction of Phe mutation at residue Trp100 increased catalytic activity compared to wild-type Gox0502 toward a set of substrates tested, which suggests that a less bulky Phe side chain at residue W100F may facilitate product release. Therefore, Gox0502 structure could provide useful information to generate desirable OYEs suitable for biotechnological applications in industry.
authors.affiliation: a State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology , East China University of Science and Technology , Shanghai , China.
authors.forename: Bo
authors.initials: B
authors.lastname: Yin
authors.forename: Jian
authors.initials: J
authors.lastname: Deng
authors.forename: Lirong
authors.initials: L
authors.lastname: Lim
authors.forename: Y Adam
authors.initials: YA
authors.lastname: Yuan
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000447:Aldehydes / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005486:Flavin Mononucleotide / Q000378:metabolism; D020600:Gluconobacter oxydans / Q000201:enzymology*; D007659:Ketones / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009252:NADPH Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D016415:Sequence Alignment; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000447:Aldehydes; D007659:Ketones; D005486:Flavin Mononucleotide; D009252:NADPH Dehydrogenase
keywords: G. oxydans Gox0502; crystal structure; old yellow enzyme; stereoselective production; structural models