Interconversion of a pair of active-site residues in Escherichia coli cystathionine gamma-synthase, E. coli cystathionine beta-lyase, and Saccharomyces cerevisiae cystathionine gamma-lyase and development of tools for the investigation of their mechanisms and reaction specificity.
Cystathionine gamma-synthase (CGS) and cystathionine beta-lyase (CBL), which comprise the transsulfuration pathway of bacteria and plants, and cystathionine gamma-lyase (CGL), the second enzyme of the fungal and animal reverse transsulfuration pathway, share approximately 30% sequence identity and are almost indistinguishable in overall structure. One difference between the active site of Escherichia coli CBL and those of E. coli CGS and Saccharomyces cerevisiae CGL is the replacement of a pair of aromatic residues, F55 and Y338, of the former by acidic residues in CGS (D45 and E325) and CGL (E48 and E333). A series of interconverting, site-directed mutants of these 2 residues was constructed in CBL (F55D, Y338E, F55D/Y338E), CGS (D45F, E325Y and D45F/E325Y) and CGL (E48A,D and E333A,D,Y) to probe the role of these residues as determinants of reaction specificity. Mutation of either position results in a reduction in catalytic efficiency, as exemplified by the 160-fold reduction in the kcat/KmL-Cys of eCGS-D45F and the 2850- and 30-fold reductions in the kcat/KmL-Cth of the eCBL-Y338E and the yCGL-E333A,Y mutants, respectively. However, the in vivo reaction specificity of the mutants was not altered, compared with the corresponding wild-type enzymes. The DeltametB and DeltametC strains, the optimized CBL and CGL assay conditions, and the efficient expression and affinity purification systems described provide the necessary tools to enable the continued exploration of the determinants of reaction specificity in the enzymes of the transsulfuration pathways.
Farsi
Ali
A
Department of Biology, Carleton University, Ottawa, ONK1S5B6, Canada.
Lodha
Pratik H
PH
Skanes
Jennifer E
JE
Los
Heidi
H
Kalidindi
Navya
N
Aitken
Susan M
SM
D020134:Catalytic Domain; D003542:Cystathionine gamma-Lyase / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology*; D005816:Genetic Complementation Test; D006639:Histidine; D007700:Kinetics; D008190:Lyases / Q000235:genetics* / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D009842:Oligopeptides; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
C471213:His-His-His-His-His-His; D050505:Mutant Proteins; D009842:Oligopeptides; D006639:Histidine; D008190:Lyases; D003542:Cystathionine gamma-Lyase; C019965:cystathionine beta-lyase