McKie, A. E. , Edlind, T., Walker, J., Mottram, J. C. and Coombs, G. H. (1998) The primitive protozoon trichomonas vaginalis contains two methionine γ-lyase genes that encode members of the γ-family of pyridoxal 5′-phosphate-dependent enzymes. Journal of Biological Chemistry, 273(10), pp. 5549-5556. (doi: 10.1074/jbc.273.10.5549) (PMID:9488680)
Full text not currently available from Enlighten.
Abstract
Methionine γ-lyase, the enzyme that catalyzes the breakdown of methionine by an α,γ-elimination reaction and is a member of the γ-family of pyridoxal 5′-phosphate-dependent enzymes, is present in high activity in the primitive protozoan parasite Trichomonas vaginalisbut is absent from mammals. Two genes, mgl1 andmgl2, encoding methionine γ-lyase, have now been isolated from T. vaginalis. They are both single copy, encode predicted proteins (MGL1 and MGL2) of 43 kDa, have 69% sequence identity with each other, and show a high degree of sequence identity to methionine γ-lyase from Pseudomonas putida (44%) and other related pyridoxal 5′-phosphate-dependent enzymes such as human cystathionine γ-lyase (42%) and Escherichia coli cystathionine β-lyase (30%). mgl1 andmgl2 have been expressed in E. coli as a fusion with a six-histidine tag and the recombinant proteins (rMGL1 and rMGL2) purified by metal-chelate affinity chromatography. rMGL1 and rMGL2 were found to have high activity toward methionine (10.4 and 0.67 μmol/min/mg of protein, respectively), homocysteine (370 and 128 μmol/min/mg of protein), cysteine (6.02 and 1.06 μmol/min/mg of protein), and O-acetylserine (3.74 and 1.51 μmol/min/mg of protein), but to be inactive toward cystathionine. Site-directed mutagenesis of an active site cysteine (C113G for MGL1 and C116G for MGL2) reduced the activity of the recombinant enzymes toward both methionine and homocysteine by approximately 80% (rMGL1) and 90% (rMGL2). In contrast, the activity of mutated rMGL2 toward cysteine andO-acetylserine was increased (to 214 and 142%, respectively), whereas that of mutated rMGL1 was reduced to 39 and 49%, respectively. These findings demonstrate the importance of this cysteine residue in the α,β-elimination and α,γ-elimination reactions catalyzed by trichomonad methionine γ-lyase.
Item Type: | Articles (Other) |
---|---|
Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Coombs, Professor Graham and Mottram, Professor Jeremy and Britten, Dr Amanda |
Authors: | McKie, A. E., Edlind, T., Walker, J., Mottram, J. C., and Coombs, G. H. |
Subjects: | Q Science > QH Natural history Q Science > QH Natural history > QH345 Biochemistry |
College/School: | College of Medical Veterinary and Life Sciences > School of Infection & Immunity College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing |
Journal Name: | Journal of Biological Chemistry |
Publisher: | American Society for Biochemistry and Molecular Biology, Inc. |
ISSN: | 0021-9258 |
ISSN (Online): | 1083-351X |
University Staff: Request a correction | Enlighten Editors: Update this record