A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis

Science

Volumn 332, Issue 6026, 2011, Pages 234-238

  Bhabha, Gira ^a   Lee, Jeeyeon ^b,c   Ekiert, Damian C ^a   Gam, Jongsik ^b   Wilson, Ian A ^a   Dyson, H Jane ^a   Benkovic, Stephen J ^b   Wright, Peter E ^a  

^a SCRIPPS RESEARCH INSTITUTE   (United States)

^b PENNSYLVANIA STATE UNIVERSITY   (United States)

^c Ajou University   (South Korea)

Author keywords

[No Author keywords available]

Indexed keywords

DIHYDROFOLATE REDUCTASE;

CATALYSIS; CATALYST; COLIFORM BACTERIUM; ENZYME ACTIVITY; PROTEIN; TIMESCALE;

ARTICLE; BACTERIUM MUTANT; CATALYSIS; CATALYST; CONFORMATIONAL TRANSITION; ENZYME ACTIVE SITE; ENZYME KINETICS; ENZYME MECHANISM; ENZYME STRUCTURE; ESCHERICHIA COLI; NONHUMAN; PRIORITY JOURNAL; STATIC ELECTRICITY; TIME;

AMINO ACID SEQUENCE; BIOCATALYSIS; CATALYTIC DOMAIN; CRYSTALLOGRAPHY, X-RAY; ESCHERICHIA COLI; FOLIC ACID; KINETICS; MODELS, MOLECULAR; MOLECULAR SEQUENCE DATA; MUTANT PROTEINS; NADP; PROTEIN CONFORMATION; TETRAHYDROFOLATE DEHYDROGENASE;

ESCHERICHIA COLI;

EID: 79953823548     PISSN: 00368075     EISSN: 10959203     Source Type: Journal    
DOI: 10.1126/science.1198542     Document Type: Article

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31. The authors acknowledge L. Tuttle for invaluable discussions, S. Bae for NMR data on the S148A mutant, H. Tien and D. Marciano of the Robotics Core at the Joint Center for Structural Genomics (JCSG) for automated crystal screening, and X. Dai for technical assistance with crystallographic data collection. This work was supported by the National Institutes of Health (NIH) grant GM75995 and the Skaggs Institute of Chemical Biology. The JCSG is supported by NIH National Institute of General Medical Sciences (NIGMS) (U54 GM094586). D.C.E. is supported by a predoctoral fellowship from the Achievement Rewards for College Scientists Foundation and grant GM080209 from the NIH Molecular Evolution Training Program. The GM/CA CAT 23-ID-D has been funded in whole or in part with federal funds from National Cancer Institute (Y1-CO-1020) and NIGMS (Y1-GM-1104). Use of the Advanced Photon Source (APS) was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under contract DE-AC02-06CH11357. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIGMS or NIH. Coordinates and structure factors of N23PP/S148A ecDHFR and lRX2-re-refined are deposited in PDB (code 3QL0 and 3QL3). G.B. and P.E.W. designed the research. G.B. performed NMR experiments. G.B. and D.C.E. performed crystallography experiments and structure refinement. I.A.W. supervised structure refinement. J.L and J.G. performed kinetic experiments and analysis. G.B., P.E.W., and S.J.B. analyzed and interpreted the data. G.B., P.E.W., and J.L wrote the manuscript. I.A.W., H.J.D., and S.J.B. edited the manuscript.