Purification and characterization of the hydantoin racemase of Pseudomonas sp. strain NS671 expressed in Escherichia coli.
The hydantoin racemase gene of Pseudomonas sp. strain NS671 had been cloned and expressed in Escherichia coli. Hydantoin racemase was purified from the cell extract of the E. coli strain by phenyl-Sepharose, DEAE-Sephacel, and Sephadex G-200 chromatographies. The purified enzyme had an apparent molecular mass of 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By gel filtration, a molecular mass of about 190 kDa was found, suggesting that the native enzyme is a hexamer. The optimal conditions for hydantoin racemase activity were pH 9.5 and a temperature of 45 degrees C. The enzyme activity was slightly stimulated by the addition of not only Mn2+ or Co2+ but also metal-chelating agents, indicating that the enzyme is not a metalloenzyme. On the other hand, Cu2+ and Zn2+ strongly inhibited the enzyme activity. Kinetic studies showed substrate inhibition, and the Vmax values for D- and L-5-(2-methylthioethyl)hydantoin were 35.2 and 79.0 mumol/min/mg of protein, respectively. The purified enzyme did not racemize 5-isopropylhydantoin, whereas the cells of E. coli expressing the enzyme are capable of racemizing it. After incubation of the purified enzyme with 5-isopropylhydantoin, the enzyme no longer showed 5-(2-methylthioethyl)hydantoin-racemizing activity. However, in the presence of 5-(2-methylthioethyl)hydantoin, the purified enzyme racemized 5-isopropylhydantoin completely, suggesting that 5-(2-methylthioethyl)hydantoin protects the enzyme from inactivation by 5-isopropylhydratoin. Thus, we examined the protective effect of various compounds and found that divalent-sulfur-containing compounds (R-S-R' and R-SH) have this protective effect.
Watabe
K
K
Odawara Research Center, Nippon Soda Co., Ltd., Kanagawa, Japan.
Ishikawa
T
T
Mukohara
Y
Y
Nakamura
H
H
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006827:Hydantoins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D019998:Racemases and Epimerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D006827:Hydantoins; D008670:Metals; D019998:Racemases and Epimerases; C074773:hydantoin racemase