Regulation of enzyme synthesis and control of activity of preexisting enzymes are two basic mechanisms for adjusting the rates of metabolic reactions in the living cell. The long-term objective of this research is to explore the molecular basis of enzyme regulation in intact cells and in isolated, purified systems. The biodegradative the ronine dehydratase, an enzyme involved in anaerobic energy metabolism and subject to multilevel controls, is an ideal prototype model system to analyze the structure and control of gene expression, and the regulatory interactions between enzymes and cellular metabolites that alter the conformation and the catalytic potential of the enzyme. In the proposed research two parallel experimental strategies-biochemical studies with purified protein and a molecular genetic analysis of the cloned DNA-will be adopted to determine the complete nucleotide sequence of the dehydratase gene, examine its control region and deduce the primary structure of the enzyme; identify the active and regulatory sites by covalent ligand binding and affinity labeling experiments to map their respective locations on the protein structure; and isolate regulatory mutants by operon-fusion technique and transposon mutagenesis to examine the factors and mechanisms involved in anaerobic enzyme induction by amino acids, cAMP, and electron acceptors. These studies will help elucidate the molecular architecture of a regulatory enzyme and its regulation by cellular metabolites. It is hoped that this new knowledge on biological control mechanisms may provide insight into the basic metabolic processes in the living cell and how loss of normal regulatory controls may trigger metabolite imbalance and biochemical disorders.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
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Chattopadhyay, S; Wu, Y; Datta, P (1997) Involvement of Fnr and ArcA in anaerobic expression of the tdc operon of Escherichia coli. J Bacteriol 179:4868-73
Wu, Y; Datta, P (1995) Influence of DNA topology on expression of the tdc operon in Escherichia coli K-12. Mol Gen Genet 247:764-7
Hagewood, B T; Ganduri, Y L; Datta, P (1994) Functional analysis of the tdcABC promoter of Escherichia coli: roles of TdcA and TdcR. J Bacteriol 176:6214-20
Ganduri, Y L; Sadda, S R; Datta, M W et al. (1993) TdcA, a transcriptional activator of the tdcABC operon of Escherichia coli, is a member of the LysR family of proteins. Mol Gen Genet 240:395-402
Wu, Y; Patil, R V; Datta, P (1992) Catabolite gene activator protein and integration host factor act in concert to regulate tdc operon expression in Escherichia coli. J Bacteriol 174:6918-27
Wu, Y F; Datta, P (1992) Integration host factor is required for positive regulation of the tdc operon of Escherichia coli. J Bacteriol 174:233-40
Schweizer, H P; Datta, P (1991) Physical linkage and transcriptional orientation of the tdc operon on the Escherichia coli chromosome. Mol Gen Genet 228:125-8
Sumantran, V N; Schweizer, H P; Datta, P (1990) A novel membrane-associated threonine permease encoded by the tdcC gene of Escherichia coli. J Bacteriol 172:4288-94
Schweizer, H P; Datta, P (1989) Identification and DNA sequence of tdcR, a positive regulatory gene of the tdc operon of Escherichia coli. Mol Gen Genet 218:516-22
Patil, R V; Datta, P (1989) Amino acid sequence of the regulatory-site glyoxylate peptide of biodegradative threonine dehydratase of Escherichia coli. J Bacteriol 171:3379-84

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