Overexpression of salicylate hydroxylase and the crucial role of lys(163) as its NADH binding site.
Expression systems for the sal gene encoding salicylate hydroxylase from Pseudomonas putida S-1 were examined and some constructs were expressed in these systems. By cultivation of Escherichia coli BL21 (DE3)/pSAH8 in LB medium at 37 degrees C with isopropyl-b-D-thiogalactopyranoside as the inducer, salicylate hydroxylase was overexpressed mainly in the form of inclusion bodies. Lower temperature cultivation at 20 degrees C after induction resulted in a large amount of the enzyme in the soluble form. The E. coli clone harboring the recombinant plasmid produced a 45 kDa protein that appeared to be electrophoretically and immunochemically identical to the P. putida enzyme and contained the same N-terminal amino acid sequence. This recombinant DNA product also exhibited properties characteristic of a flavoprotein and was fully functional as salicylate hydroxylase. Based on chemical modification of the salicylate hydroxylase from P. putida, Lys163 was previously proposed to be the NADH binding site. In this study, to obtain a better understanding of the predicted role of Lys163, this residue in the active center of salicylate hydroxylase was replaced with Arg, Gly, or Glu by conventional site-directed mutagenesis. Kinetic studies using these mutant enzymes and the recombinant enzyme revealed increases in apparent K(m) values for NADH in the order of wild-type enzyme > K163R > K163G > K163E, with some decreases in V(max). Examination of the recombinant enzyme and K163G indicated that the pH dependency of K(m) on NADH with pK(a) 10.5 is lost by mutation despite the lack of changes in V(max) values, suggesting a requirement for the lysine residue as the NADH binding site. Based on these results, Lys163 is proposed to play a role in the binding of NADH at the active site through an ionic bond rather than playing a role in catalysis.
Suzuki
K
K
Department of Chemistry, Faculty of Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. kenzi@cacheibm.s.kanazawa-u.ac.jp
Asao
E
E
Nakamura
Y
Y
Nakamura
M
M
Ohnishi
K
K
Fukuda
S
S
D019943:Amino Acid Substitution; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D016958:Pseudomonas putida / Q000737:chemistry
D001051:Apoenzymes; D009243:NAD; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase; D008239:Lysine