EFFECT OF MUTATIONS IN THE TRANSMETHYLASE AND DEHYDROGENASE CHELATASEDOMAINS OF SIROHAEM SYNTHASE (CYSG) ON SIROHAEM AND COBALAMIN BIOSYNTHESIS/

The Escherichia coli CysG protein (sirohaem synthase) catalyses four s eparate reactions that are required for the transformation of uroporph yrinogen III into sirohaem, initially two S-adenosyl-L-methionine-depe ndent transmethylations at positions 2 and 7, mediated through the C-t erminal, or CysG(A), catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal , or CysG(B), catalytic domain of the enzyme. This report describes ho w the deletion of the NAD(+)-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysG(B) but does not affect the catalytic activity of CysG(A), wherea s the mutation of a number of phylogenetically conserved residues with in CysG(A) is detrimental to the transmethylation reaction but does no t affect the activity of CysG(B). Further studies have shown that CysG (B) is not essential for cobalamin biosynthesis because the presence o f the Salmonella typhimurium CobI operon with either cysG(A) or the Ps eudomonas denitrificans cobA are sufficient for the synthesis of cobyr ic acid in an E. coli cysG deletion strain. Evidence is also presented to suggest that a gene within the S. typhimurium CobI operon might ac t as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.