The research involves studies of the mechanisms of the reactions catalyzed by the adenylate cyclase from Salmonella typhimurium and three enzymes involved in dark DNA repair processes in Escherichia coli. The bacterial adenylate cyclase is intimately involved in the process of catabolite repression, and we have demonstrated that this enzyme contains a discrete catalytic domain. We will use recombinant DNA techniques to facilitate purification of the intact enzyme as well as the catalytic domain; with the purified enzymes we will undertake kinetic and enzymological studies of the reaction mechanism. Dark DNA repair involves the correction of chemically produced lesions in DNA; we will focus our attention on the mechanisms of three enzymes that are involved in these repair processes: 1) Uracil-DNA glycosylase catalyzes the hydrolysis of the glycosidic bonds in deoxyuridine residues that arise by the spontaneous hydrolysis of the 4-amino group of cytosine. Our studies will employ sequence defined synthetic oligonucleotides as substrates, and we will use kinetic isotope effect studies to ascertain whether catalysis involves the formation of a covalent adduct with the uridine residue; we will also use primer directed mutagenesis to ascertain the importance of an essential cysteine residue in catalysis. 2) Bacteriophage T4 UV endonuclease V initiates the repair of apurinic/apyrimidinic (AP) sites in DNA (such as those produced by uracil-DNA glycosylase) by cleavage of the phosphodiester backbone on the 3'-side of the AP site. Our studies will employ sequence defined synthetic oli8gonucleotides as substrates, and we will use NMR methods to identify the reaction products. Our work on this enzyme will also involve recombinant DNA techniques to facilitate purification of large amounts of the enzyme. 3) 06 Methylguanine-DNA methyltransferase removes mutagenic alkyl groups from the 06 position of guanine, thereby preventing base pair transversions. This reaction involves the irreversible transfer of the methyl group to a cysteine residue in the methyltransferase, and we will determine the stereochemical course of this reaction using sequence defined synthetic oligonucleotides as substrates; the required configurational analyses will be accomplished by 1H NMR spectroscopy.

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National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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University of Maryland College Park
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College Park
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Landro, J A; Gerlt, J A; Kozarich, J W et al. (1994) The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: mechanistic and crystallographic evidence for stereospecific alkylation by (R)-alpha-phenylglycidate. Biochemistry 33:635-43
Mitra, B; Gerlt, J A; Babbitt, P C et al. (1993) A novel structural basis for membrane association of a protein: construction of a chimeric soluble mutant of (S)-mandelate dehydrogenase from Pseudomonas putida. Biochemistry 32:12959-67
Petsko, G A; Kenyon, G L; Gerlt, J A et al. (1993) On the origin of enzymatic species. Trends Biochem Sci 18:372-6
Gerlt, J A; Gassman, P G (1993) Understanding the rates of certain enzyme-catalyzed reactions: proton abstraction from carbon acids, acyl-transfer reactions, and displacement reactions of phosphodiesters. Biochemistry 32:11943-52
Mazumder, A; Gerlt, J A; Absalon, M J et al. (1991) Stereochemical studies of the beta-elimination reactions at aldehydic abasic sites in DNA: endonuclease III from Escherichia coli, sodium hydroxide, and Lys-Trp-Lys. Biochemistry 30:1119-26
Powers, V M; Koo, C W; Kenyon, G L et al. (1991) Mechanism of the reaction catalyzed by mandelate racemase. 1. Chemical and kinetic evidence for a two-base mechanism. Biochemistry 30:9255-63
Landro, J A; Kallarakal, A T; Ransom, S C et al. (1991) Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant. Biochemistry 30:9274-81
Tsou, A Y; Ransom, S C; Gerlt, J A et al. (1990) Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli. Biochemistry 29:9856-62
Tsou, A Y; Ransom, S C; Gerlt, J A et al. (1989) Selection and characterization of a mutant of the cloned gene for mandelate racemase that confers resistance to an affinity label by greatly enhanced production of enzyme. Biochemistry 28:969-75
Holland, M M; Leib, T K; Gerlt, J A (1988) Isolation and characterization of a small catalytic domain released from the adenylate cyclase from Escherichia coli by digestion with trypsin. J Biol Chem 263:14661-8

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