title: X-ray crystallographic and enzymatic analyses of shikimate dehydrogenase from Staphylococcus epidermidis.
abstract: Shikimate dehydrogenase (SDH) catalyzes the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway. In this study, we determined the kinetic properties and crystal structures of Staphylococcus epidermidis SDH (SeSDH) both in its ligand-free form and in complex with shikimate. SeSDH has a k(cat) of 22.8 s(-1) and a K(m) of 73 mum towards shikimate, and a K(m) of 100 microM towards NADP. The overall folding of SeSDH comprises the N-terminal alpha/beta domain for substrate binding and the C-terminal Rossmann fold for NADP binding. The active site is within a large groove between the two domains. Residue Tyr211, normally regarded as important for substrate binding, does not interact with shikimate in the binary SeSDH-shikimate complex structure. However, the Y211F mutation leads to a significant decrease in k(cat) and a minor increase in the K(m) for shikimate. The results indicate that the main function of Tyr211 may be to stabilize the catalytic intermediate during catalysis. The NADP-binding domain of SeSDH is less conserved. The usually long helix specifically recognizing the adenine ribose phosphate is substituted with a short 3(10) helix in the NADP-binding domain. Moreover, the interdomain angle of SeSDH is the widest among all known SDH structures, indicating an inactive 'open' state of the SeSDH structure. Thus, a 'closing' process might occur upon NADP binding to bring the cofactor close to the substrate for catalysis.
authors.affiliation: Institutes of Biomedical Sciences and Key Laboratory of Medical Molecular Virology, Institute of Medical Microbiology, Shanghai Medical College, Fudan University, China.
authors.forename: Cong
authors.initials: C
authors.lastname: Han
authors.forename: Tiancen
authors.initials: T
authors.lastname: Hu
authors.forename: Dalei
authors.initials: D
authors.lastname: Wu
authors.forename: Su
authors.initials: S
authors.lastname: Qu
authors.forename: Jiahai
authors.initials: J
authors.lastname: Zhou
authors.forename: Jianping
authors.initials: J
authors.lastname: Ding
authors.forename: Xu
authors.initials: X
authors.lastname: Shen
authors.forename: Di
authors.initials: D
authors.lastname: Qu
authors.forename: Hualiang
authors.initials: H
authors.lastname: Jiang
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009249:NADP / Q000378:metabolism; D017510:Protein Folding; D012765:Shikimic Acid / Q000378:metabolism; D013212:Staphylococcus epidermidis / Q000201:enzymology*
chemical_list: D012765:Shikimic Acid; D009249:NADP; D000429:Alcohol Oxidoreductases; C022641:Shikimate dehydrogenase