Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein.
The C-terminus of the Escherichia coli CysG protein, consisting of amino acids 202-457, was expressed as a recombinant protein using gene dissection methodology. Analysis of the activity of this truncated protein, termed CysGA, revealed that it was able to methylate uroporphyrinogen III in the same S-adenosyl-L-methionine (SAM)-dependent manner as the complete CysG protein. However, this truncated protein was not able to complement E. coli cysG cells, thereby suggesting that the first 201 amino acids of the CysG protein had an enzymic activity associated with the conversion of dihydrosirohydrochlorin into sirohaem. Analysis of the N-terminus of the CysG protein revealed the presence of a putative pyridine dinucleotide binding site. When the purified CysG protein was incubated with NADP+, uroporphyrinogen III and SAM the enzyme was found to catalyse a coenzyme-mediated dehydrogenation to form sirohydrochlorin. The CysGA protein on the other hand showed no such coenzyme-dependent activity. Analysis of the porphyrinoid material isolated from strains harbouring plasmids containing the complete and truncated cysG genes suggested that the CysG protein was also involved in ferrochelation. The evidence presented in this paper suggests that the CysG protein is a multifunctional protein involved in SAM-dependent methylation, pyridine dinucleotide dependent dehydrogenation and ferrochelation.
Warren
M J
MJ
School of Biological Sciences, Queen Mary and Westfield College, London, U.K.
Bolt
E L
EL
Roessner
C A
CA
Scott
A I
AI
Spencer
J B
JB
Woodcock
S C
SC
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D006418:Heme / Q000096:biosynthesis; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine / Q000378:metabolism; D014577:Uroporphyrinogens / Q000378:metabolism; D014578:Uroporphyrins / Q000378:metabolism
D011994:Recombinant Proteins; D014577:Uroporphyrinogens; D014578:Uroporphyrins; D006418:Heme; D009249:NADP; D012436:S-Adenosylmethionine; C063209:15,23-dihydrosirohydrochlorin; D008780:Methyltransferases; C060508:uroporphyrin-III C-methyltransferase