Purification and characterization of Thermotoga maritima homoserine transsuccinylase indicates it is a transacetylase.
The methionine biosynthetic pathway found in bacteria is controlled at the first step, acylation of the gamma-hydroxyl of homoserine. This reaction is catalyzed by one of two unique enzymes, homoserine transacetylase or homoserine transsuccinylase, which have no amino acid sequence similarity. We cloned, expressed, and purified homoserine transsuccinylase from the thermophilic bacterium Thermotoga maritima. Substrate specificity experiments demonstrated that acetyl-coenzyme A (CoA) is the preferred acyl donor and is used at least 30-fold more efficiently than succinyl-CoA. Steady-state kinetic experiments confirm that the enzyme utilizes a ping-pong kinetic mechanism in which the acetate group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine. The maximal velocity, V/K (acetyl-CoA) and V/K (homoserine), all exhibited bell-shaped pH curves with apparent pKs of 6.0-6.9 and 8.2-8.8. The enzyme was inactivated by iodoacetamide in a pH-dependent manner, with an apparent pK of 6.3, suggesting the presence of an active-site cysteine residue which forms an acetyl-enzyme thioester intermediate during catalytic turnover, similar to observations with other transsuccinylases. In addition, the enzyme is highly stable at elevated temperatures, maintaining full activity at 70 degrees C. Taken together, these data suggest that the T. maritima enzyme functions biochemically as a transacetylase, despite having the sequence of a transsuccinylase.
Goudarzi
Maryam
M
Department of Chemistry and Biochemistry, George Mason University, 10900 University Boulevard, Manassas, VA 20110, USA.
Born
Timothy L
TL
D000123:Acetyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D017666:Deuterium Oxide; D004926:Escherichia coli / Q000201:enzymology; D006193:Haemophilus influenzae / Q000201:enzymology; D051082:Homoserine O-Succinyltransferase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008715:Methionine / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012997:Solvents; D013045:Species Specificity; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D014867:Water
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D012997:Solvents; D014867:Water; D008715:Methionine; D000123:Acetyltransferases; C021369:homoserine O-acetyltransferase; D051082:Homoserine O-Succinyltransferase; D017666:Deuterium Oxide