Investigating a catalytic mechanism of hyperthermophilic L-threonine dehydrogenase from Pyrococcus horikoshii.
Based on our first structural data of L-threonine dehydrogenase (TDH) of Pyrococcus horikoshii (PhTDH), we examined its catalytic mechanism. The structural analysis indicated that a catalytic zinc atom at the active centre of PhTDH is coordinated by four residues (Cys42, His67, Glu68 and Glu152) with low affinity. These residues are highly conserved in alcohol dehydrogenases (ADHs) and TDHs. Several PhTDH mutants were prepared with respect to Glu152 and other residues, relating to the proton relay system that is substantially a rate-limiting step in ADH. It was found that the E152D mutant showed 3-fold higher turnover rate and reduced affinities toward L-threonine and NAD(+), compared to wild-type PhTDH. The kinetic analysis of Glu152 mutants indicated that the carboxyl group of Glu152 is important for expressing the catalytic activity. The results obtained from pH dependency of kinetic parameters suggested that Glu152 to Asp substitution causes the enhancement of deprotonation of His47 or ionization of zinc-bound water and threonine in the enzyme-NAD(+) complex. Furthermore, it was predicted that the access of threonine substrate to the enzyme-NAD(+) complex induces a large conformational change in the active domain of PhTDH. From these results, we propose here that the proton relay system works as a catalytic mechanism of PhTDH.
Higashi
Noriko
N
Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan.
Tanimoto
Koichi
K
Nishioka
Motomu
M
Ishikawa
Kazuhiko
K
Taya
Masahito
M
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry
D019843:Archaeal Proteins; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase; D015032:Zinc