Biochemical analysis on the parallel pathways of methionine biosynthesis in Corynebacterium glutamicum.
Two alternative pathways for methionine biosynthesis are known in Corynebacterium glutamicum: one involving transsulfuration (mediated by metB and metC) and the other involving direct sulthydrylation (mediated by metY). In this study, MetB (cystathionine gamma-synthase) and MetY (O-acetylhomoserine sulfhydrylase) from C. glutamicum were purified to homogeneity and the biochemical parameters were compared to assess the functional and evolutionary importance of each pathway. The molecular masses of the native MetB and MetY proteins were measured to be approximately 170 and 280 kDa, respectively, showing that MetB was a homotetramer of 40-kDa subunits and MetY was a homohexamer of 45-kDa subunits. The Km values for the O-acetylhomoserine catalysis effected by MetB and MetY were 3.9 and 6.4 mM, and the maximum catalysis rates were 7.4 (kcat = 21 s(-1)) and 6.0 (kcat=28 s(-1)) micromol mg(-1) min(-1), respectively. This suggests that both MetB and MetY can be comparably active in vivo. Nevertheless, the Km value for sulfide ions by MetY was 8.6 mM, which was too high, considering the physiological condition. Moreover, MetB was active at a broad range of temperatures (30 and 65 degrees C) and pH (6.5 and 10.0), as compared with MetY, which was active in a range from 30 to 45 degrees C and at pH values from 7.0 to 8.5. In addition, MetY was inhibited by methionine, but MetB was not. These biochemical data may provide insight on the role of the parallel pathways of methionine biosynthesis in C. glutamicum with regard to cell physiology and evolution.
Hwang
Byung-Joon
BJ
Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea.
Park
Soo-Dong
SD
Kim
Younhee
Y
Kim
Pil
P
Lee
Heung-Shick
HS
D019757:Carbon-Oxygen Lyases / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D048230:Corynebacterium glutamicum / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000096:biosynthesis*; D013696:Temperature
D008715:Methionine; C018725:O-acetylhomoserine (thiol)-lyase; C022696:O-succinylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases