Cloning and overexpression of the oah1 gene encoding O-acetyl-L-homoserine sulfhydrylase of Thermus thermophilus HB8 and characterization of the gene product.
The oah1 gene of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned, sequenced, and overexpressed in Escherichia coli cells. The gene product having a high O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity was purified to homogeneity, with a recovery of approximately 40% and a purification ratio of 81-fold, both calculated from the cell-homogenate. The protein showed molecular masses of approximately 163000 (for the native form) and 47000 (for the subunit). The isoelectric point was pH 6.0. The optimum temperature and pH for the activity were approximately 70 degrees C and pH 7.8, respectively. The enzyme was also shown to be very stable at high temperature (90% activity remaining at 90 degrees C for 60 min at pH 7.8) and in a wide range of pH (pH 4-12 at room temperature). The absorption spectrum showed a peak at 425 nm, and hydroxylamine hydrochloride (0.1 mM) inhibited approximately 90% of the activity, suggesting formation of a Schiff base with pyridoxal 5'-phosphate. The enzyme showed an apparent K(m) value of 6.8 mM for O-acetyl-L-homoserine, a V(max) value of 165 micromol/min per mg of protein at a fixed sulfide concentration of 5 mM, and also an apparent K(m) value of approximately 1.3 mM for sulfide (with 25 mM acetylhomoserine). L-Methionine (1 mM) inhibited the enzyme activity by 67%. Based on these findings, it was discussed that this enzyme might be inactive under ordinary conditions but might become active as an alternative homocysteine synthase in T. thermophilus HB8, only under such conditions as deficiency in transsulfuration, bringing about a sufficient amount of sulfide available in the cell.
Shimizu
H
H
Department of Biotechnology, Faculty of Agriculture, Gifu University, Japan.
Yamagata
S
S
Masui
R
R
Inoue
Y
Y
Shibata
T
T
Yokoyama
S
S
Kuramitsu
S
S
Iwama
T
T
D019757:Carbon-Oxygen Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D002384:Catalysis; D003001:Cloning, Molecular; D003547:Cysteine Synthase; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics*
D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D003547:Cysteine Synthase; C103381:MET17 protein, S cerevisiae; C018725:O-acetylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases