Cloning and characterization of the genes for the two homocysteine transmethylases of Escherichia coli.

Abstract

We have cloned the genes for the two homocysteine transmethylases of Escherichia coli K12. The vitamin B12-independent enzyme is encoded by the metE gene while the metH gene codes for the vitamin B12-requiring enzyme. Overexpression of the gene products and Tn1000 mutagenesis have enabled the metE and metH gene products to be identified as 99 kDa and 130 kDa polypeptides, respectively. The truncated polypeptides generated by Tn1000 insertion were used to determine the direction of transcription of the metE and metH genes. Negative complementation suggests that the MetH enzyme exists as an oligomer. Investigation of the expression of the chromosomal- and plasmid-encoded gene products confirms that metE is subject to negative control by vitamin B12 and methionine, and that metH is under positive control by the cofactor and negative control by methionine. For vitamin B12 and methionine to act as regulatory effectors in metE control, functional metH and metJ genes are required, respectively. The use of stable Tn1000-generated fragments of the metE product as electrophoretic markers for the plasmid-encoded metE gene product demonstrated that the two regulatory proteins involved in negative control of metE are present in excess. Under conditions whereby both forms of negative metE control are non-functional, the metE gene product represented about 90% of the total protein, and cell growth was severely impaired.