3-methyladenine-DNA Glycosylase I from Escherichia coli - Computer Modeling and Supporting Experimental Evidence

Abstract

Obesity is an increasing health problem in affluent societies that contributes to the prevalence of serious health diseases such as diabetes and heart disease. Current pharmacotherapies for obesity have side effects and are not effective in the long term, therefore, opportunities exist to develop novel therapeutics to treat this disorder. Peptides derived from the C-terminal domain of human growth hormone have shown to have anti-lipogenic and lipolytic activity. Growth hormone is a multifunctional protein (22-26 kDa) comprised of discrete bioactive domains responsible for several biological actions including the reduction and redistribution of body fat (Pullin et al., 1981) and the promotion of growth. Growth hormone is synthesized in the pituitary gland and stored in the cells of the anterior pituitary (Goodman et al., 1969). In mature animals it is released from these cells in response to insulin-induced hypoglycemia, starvation, amino acid infusion and neural stimuli including emotional stress. These responses suggest that growth hormone may play a role in the regulation of metabolism of energy in adult animals.AOD9401 is a cyclic 15 amino acid peptide containing a disulphide bond derived from human growth hormone. Biological investigations have revealed that AOD9401 has lipolytic and anti-lipogenic actions on rodent, porcine and human adipose tissues. Moreover, the biological activity of AOD9401 has been improved by substitution of key amino acids or functional side chains. These active mutant AOD9401 analogues show great promise for the development of new and more potent anti-obesity peptides. We are investigating whether the lipolytic and antilipogenic activity of AOD9401 and analogues are related to a specific molecular conformation. A conformational analysis of the peptides will be presented.Escherichia coli possesses two different N-methylpurine-D N A glycosylases. 3-methyladenine-DNA glycosylase I1 induced as a one of enzymes of adaptive response to alkylating agents with broad substrate specificity. 3-methyladenine-D N A glycosylase I (TagA protein) is expressed constitutively and excises only 3-methyladenine and 3-methylguanine from alkylated DNA. The structure of this enzyme has not yet been determined experimentally. We propose a tree-dimensional model of the TagA protein based on the threading algorithm. The model shows that TagA is mostly a a-helical protein, in agreement with circular dichroism measurements.None of the eight cysteines present in the TagA sequence forms a disulfide bridge in the model structure, which has also been experimentally verified with the use of Ellman method. Investigation of functional aspects of the N-terminal region ofEscherichia coli T-protein of the glycine cleavage system The glycine cleavage system is composed of four proteins named P-, H-, T-, and L-protein. T-protein is a folate-dependent enzyme and catalyzes the formation of NH) and CH2-THF from the aminomethyl intermediate attached to H-protein after the decarboxylation of glycine. A mutant with N-terminal7-r...