The members of the mechanistically diverse enolase superfamily share a bidomain structure in which a capping domain formed by the N- and C-termini of the polypeptide determines substrate specificity and the functional groups at the C-terminal end of a (beta/alpha)7beta-barrel domain determine reaction mechanism. We want to understand how the structure of the barrel delivers different functions, allowing us to use that information to 1) assist prediction of the functions of unknowns proteins discovered in genome sequencing projects; and2) redesign active sites to catalyze """"""""new"""""""" reactions. The project involves four Specific Aims that integrate mechanistic and structural studies. The mechanistic studies will be performed in Dr. Gerlt's laboratory at Illinois (P.I.); the structural studies will be performed in Dr. Rayment's laboratory at Wisconsin (Co-P.I.):1) Structure/function relationships will be established for the newly assigned D-gluconate dehydratases, L-rhamnonate dehydratases, and D-altronate dehydratases in which the active site motifs differ from those previously identified for other dehydratases.2) Functions will be assigned to unknown members by screening purified proteins from several microbial species encoding multiple members for acid sugar dehydratasetisomerase activities.3) Structure/function relationships will be established for o-succinylbenzoate synthases, L-Ala-D/L-Gluepimerases, D-galactonate dehydratases, and D-glucarate dehydratases in which the active site motifs differfrom those previously characterized for """"""""orthologues"""""""" that catalyze the same reactions.4) We will test a structural blueprint for functional diversity in the (beta/alpha)7beta-barrel fold by determining whether new functions can be generated by in vitro evolution.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052594-12
Application #
7100885
Study Section
Biochemistry Study Section (BIO)
Program Officer
Ikeda, Richard A
Project Start
1995-08-02
Project End
2008-07-31
Budget Start
2006-08-01
Budget End
2008-07-31
Support Year
12
Fiscal Year
2006
Total Cost
$442,890
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Gerlt, John A; Babbitt, Patricia C (2009) Enzyme (re)design: lessons from natural evolution and computation. Curr Opin Chem Biol 13:10-8
Rakus, John F; Fedorov, Alexander A; Fedorov, Elena V et al. (2008) Evolution of enzymatic activities in the enolase superfamily: L-rhamnonate dehydratase. Biochemistry 47:9944-54
Vick, Jacob E; Gerlt, John A (2007) Evolutionary potential of (beta/alpha)8-barrels: stepwise evolution of a ""new"" reaction in the enolase superfamily. Biochemistry 46:14589-97
Rakus, John F; Fedorov, Alexander A; Fedorov, Elena V et al. (2007) Evolution of enzymatic activities in the enolase superfamily: D-Mannonate dehydratase from Novosphingobium aromaticivorans. Biochemistry 46:12896-908
Glasner, Margaret E; Gerlt, John A; Babbitt, Patricia C (2007) Mechanisms of protein evolution and their application to protein engineering. Adv Enzymol Relat Areas Mol Biol 75:193-239, xii-xiii
Glasner, Margaret E; Gerlt, John A; Babbitt, Patricia C (2006) Evolution of enzyme superfamilies. Curr Opin Chem Biol 10:492-7
Brown, Shoshana D; Gerlt, John A; Seffernick, Jennifer L et al. (2006) A gold standard set of mechanistically diverse enzyme superfamilies. Genome Biol 7:R8
Yew, Wen Shan; Fedorov, Alexander A; Fedorov, Elena V et al. (2006) Evolution of enzymatic activities in the enolase superfamily: D-tartrate dehydratase from Bradyrhizobium japonicum. Biochemistry 45:14598-608
Yew, Wen Shan; Fedorov, Alexander A; Fedorov, Elena V et al. (2006) Evolution of enzymatic activities in the enolase superfamily: L-fuconate dehydratase from Xanthomonas campestris. Biochemistry 45:14582-97
Sakai, Ayano; Xiang, Dao Feng; Xu, Chengfu et al. (2006) Evolution of enzymatic activities in the enolase superfamily: N-succinylamino acid racemase and a new pathway for the irreversible conversion of D- to L-amino acids. Biochemistry 45:4455-62

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