Abstract
The methionine biosynthetic pathway found in bacteria is controlled at the first step, acylation of the γ-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°C. Taken together, these data suggest that the T. maritima enzyme functions biochemically as a transacetylase, despite having the sequence of a transsuccinylase.
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Abbreviations
- EcHTS :
-
Escherichia coli homoserine transsuccinylase
- HiHTA :
-
Haemophilus influenzae homoserine transacetylase;
- HTA :
-
Homoserine transacetylase
- HTS :
-
Homoserine transsuccinylase
- IAA :
-
Iodoacetamide
- OAH :
-
O-acetylhomoserine
- OSH :
-
O-succinylhomoserine
- PLP :
-
Pyridoxal phosphate
- SAM :
-
S-adenosylmethionine
- SDS-PAGE :
-
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- SELDI-TOF MS :
-
Surface-enhanced laser desorption/ionization-time of flight mass spectrometry
- TEA :
-
Triethanolamine
- TmHTA :
-
Thermotoga maritima homoserine transacetylase
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Acknowledgements
This work was supported by the National Institutes of Health (GM064513 to TLB). The authors would like to thank Katharine Ziegler for the critical reading of this manuscript and her assistance in the experimental procedures.
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Communicated by G. Antranikian
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Goudarzi, M., Born, T.L. Purification and characterization of Thermotoga maritima homoserine transsuccinylase indicates it is a transacetylase. Extremophiles 10, 469–478 (2006). https://doi.org/10.1007/s00792-006-0522-3
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DOI: https://doi.org/10.1007/s00792-006-0522-3