The goal of this project is to determine the molecular structure of the adaptive landscapes across which two enzymes evolve. By relating enzyme structure to enzyme function, and enzyme function to fitness, this work will provide a detailed understanding of the causes of adaptation and role of constraint in biochemical evolution. The NADP-dependent isocitrate dehydrogenase (IDH) and NAD-dependent isopropylmalate dehydrogenase (IMDH) of Escherichia coil provide an ideal experimental system to explore these relations. Both enzymes belong to an ancient and extensive superfamily, the phylogeny of which provides evolutionary history. Their roles in metabolism are thoroughly understood, IMDH in leucine biosynthesis and IDH in Krebs' cycle. Their kinetic, catalytic and regulatory mechanisms (IMDH at the transcriptional level, IDH by post-translational phosphorylation) have been determined. Native, mutant, and modified enzymes, with and without substrates bound. It had been subject to detailed X-ray crystallographic studies This rich and detailed background provides the necessary basis for understanding adaptation and constraint in molecular evolution. Phylogenetic analyses reveal that 3.5 billion years ago an ancient bacterial NAD-dependent IDH evolved the ability to utilize NADP. In contrast, all known IMDHs utilize NAD. Protein engineering has confirmed that only 6 out of 250 amino acid replacements determine which coenzyme is used. With so few sites determining coenzyme usage so all possible genetic intermediates between the two extreme phenotypes can be constructed. Competition between strains of Escherichia coil carrying different mutant alleles will be used to determine the fitness. Thus, the relations between catalytic efficiency, substrate specificity and fitness will be rigorously determined, enabling the molecular basis of the adaptive shift in coenzyme utilization by IDH (for growth on acetate), and the constraints that force IMDH to use NAD (enzymes with intermediate phenotypes are less fit) to be understood in terms of adaptive landscapes. By investigating what has, and has not, happened during 4 billion years of molecular evolutionary history will not only enrich our understanding of biochemical adaptation, but may also provide subtle insights into the relations between protein structure and function, ones that might be overlooked by more traditional approaches. Many of these may prove helpful to the rational design of catalysts for industry, and of drugs for medicine.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM060611-01A2
Application #
6317483
Study Section
Genetics Study Section (GEN)
Program Officer
Preusch, Peter C
Project Start
2001-06-01
Project End
2005-05-31
Budget Start
2001-06-01
Budget End
2002-05-31
Support Year
1
Fiscal Year
2001
Total Cost
$278,076
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Gonçalves, Susana; Miller, Stephen P; Carrondo, Maria A et al. (2012) Induced fit and the catalytic mechanism of isocitrate dehydrogenase. Biochemistry 51:7098-115
Lunzer, Mark; Golding, G Brian; Dean, Antony M (2010) Pervasive cryptic epistasis in molecular evolution. PLoS Genet 6:e1001162
Stoebel, Daniel M; Dean, Antony M; Dykhuizen, Daniel E (2008) The cost of expression of Escherichia coli lac operon proteins is in the process, not in the products. Genetics 178:1653-60
Merlo, Lauren M F; Lunzer, Mark; Dean, Antony M (2007) An empirical test of the concomitantly variable codon hypothesis. Proc Natl Acad Sci U S A 104:10938-43
Dean, Antony M; Thornton, Joseph W (2007) Mechanistic approaches to the study of evolution: the functional synthesis. Nat Rev Genet 8:675-88
Miller, Stephen P; Lunzer, Mark; Dean, Antony M (2006) Direct demonstration of an adaptive constraint. Science 314:458-61
Lunzer, Mark; Miller, Stephen P; Felsheim, Roderick et al. (2005) The biochemical architecture of an ancient adaptive landscape. Science 310:499-501
Zhu, Guoping; Golding, G Brian; Dean, Antony M (2005) The selective cause of an ancient adaptation. Science 307:1279-82