ACC synthase catalyzes the conversion of S- adenosylmethionine (SAM) to amino cyclopropane carboxylate (ACC). ACC is the immediate precursor to ethylene, the plant hormone responsible for, inter alia, wound healing, fruit ripening and senescence. We plan to solve X-ray structures and biochemically characterize SAM-analog complexes with wild type and mutant enzymes in order to understand the special chemistry leading to the formation of the unique cyclopropane ring. While rational enzyme design has enjoyed some success, directed evolution has opened up the possibility of discovering mutations far from the catalytic site of an enzyme that effect both catalytic rates and reaction specificity. This novel technology will be adopted to convert aspartate aminotransferase to tyrosine aminotransferase and ACC synthase to a SAM aminotransferase. The functions of two genes (yjiR and ydcR), which code for two previously unknown pyridoxal phosphate (PLP) binding proteins, will be studied by a combination of bioinformatics, biochemical and nutritional microbiology techniques. The one known SAM aminotransferase, diaminopelargonic acid (DAPA) synthase from the biotin synthetic pathway, will be investigated to try to understand how two enzymes process the same substrate to different products-ACC synthase directs SAM to ACC while DAPA synthase converts it to DAPA. Mutations in cystathionine-beta-synthase are frequently responsible for human homocystinuria. The mechanism of action of this PLP-dependent enzyme will be pursued with particular reference to try to understand how the mutations contribute to the pathology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM035393-17
Application #
6371029
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Jones, Warren
Project Start
1985-07-01
Project End
2005-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
17
Fiscal Year
2001
Total Cost
$387,585
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Muratore, Kathryn E; Engelhardt, Barbara E; Srouji, John R et al. (2013) Molecular function prediction for a family exhibiting evolutionary tendencies toward substrate specificity swapping: recurrence of tyrosine aminotransferase activity in the I? subfamily. Proteins 81:1593-609
Shultzaberger, Ryan K; Maerkl, Sebastian J; Kirsch, Jack F et al. (2012) Probing the informational and regulatory plasticity of a transcription factor DNA-binding domain. PLoS Genet 8:e1002614
Deu, Edgar; Kirsch, Jack F (2011) Engineering homooligomeric proteins to detect weak intersite allosteric communication: aminotransferases, a case study. Protein Sci 20:1991-2003
Hanes, Melinda S; Reynolds, Kimberly A; McNamara, Case et al. (2011) Specificity and cooperativity at ?-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions. Proteins 79:1267-76
Sankararaman, Sriram; Sha, Fei; Kirsch, Jack F et al. (2010) Active site prediction using evolutionary and structural information. Bioinformatics 26:617-24
Deu, Edgar; Dhoot, Jashdeep; Kirsch, Jack F (2009) The partially folded homodimeric intermediate of Escherichia coli aspartate aminotransferase contains a ""molten interface"" structure. Biochemistry 48:433-41
Muratore, Kathryn E; Srouji, John R; Chow, Margaret A et al. (2008) Recombinant expression of twelve evolutionarily diverse subfamily Ialpha aminotransferases. Protein Expr Purif 57:34-44
Yin, Yifeng; Kirsch, Jack F (2007) Identification of functional paralog shift mutations: conversion of Escherichia coli malate dehydrogenase to a lactate dehydrogenase. Proc Natl Acad Sci U S A 104:17353-7
Deu, Edgar; Kirsch, Jack F (2007) Cofactor-directed reversible denaturation pathways: the cofactor-stabilized Escherichia coli aspartate aminotransferase homodimer unfolds through a pathway that differs from that of the apoenzyme. Biochemistry 46:5819-29
Reynolds, Kimberly A; Thomson, Jodi M; Corbett, Kevin D et al. (2006) Structural and computational characterization of the SHV-1 beta-lactamase-beta-lactamase inhibitor protein interface. J Biol Chem 281:26745-53

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