Probing the active site of homoserine trans-succinylase.
Homoserine trans-succinylase is the first enzyme in methionine biosynthesis of Escherichia coli and catalyzes the activation of homoserine via a succinylation reaction. The in vivo activity of this enzyme is subject to tight regulation by several mechanisms, including repression and activation of gene expression, feedback inhibition, temperature regulation and proteolysis. This complex regulation reflects the key role of this enzyme in bacterial metabolism. Here, we demonstrate--using proteomics and high-resolution mass spectrometry--that succinyl is covalently bound to one of the two adjacent lysine residues at positions 45 and 46. Replacing these lysine residues by alanine abolished the enzymatic activity. These findings position the lysine residues, one of which is conserved, at the active site.
Rosen
Ran
R
Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.
Becher
Dörte
D
Büttner
Knut
K
Biran
Dvora
D
Hecker
Michael
M
Ron
Eliora Z
EZ
D000217:Acyltransferases / Q000737:chemistry*; D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D015964:Gene Expression Regulation, Bacterial; D006714:Homoserine / Q000378:metabolism*; D051082:Homoserine O-Succinyltransferase; D007525:Isoelectric Focusing; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D008715:Methionine / Q000096:biosynthesis; D040901:Proteomics; D017422:Sequence Analysis, DNA
D029968:Escherichia coli Proteins; D006714:Homoserine; D008715:Methionine; D000217:Acyltransferases; D051082:Homoserine O-Succinyltransferase; D008239:Lysine; D000409:Alanine