Characterization of the 6-aminohexanoate-dimer hydrolase from Pseudomonas sp. NK87.
The DNA base sequence of the Pseudomonas sp. NK87 gene (P-nylB) for 6-aminohexanoate-dimer hydrolase (P-EII), a xenobiotic-compound-degrading enzyme, was determined. It has an open reading frame of 1188 bp, initiated by ATG and terminated by TAG, and coding for 396 amino acids. The base sequence of the open reading frame has 53% sequence similarity to that of the gene for the same enzyme of Flavobacterium sp. KI72 (F-nylB) and 35% sequence similarity with respect to the deduced amino acid sequence. The P-EII enzyme was purified from an Escherichia coli clone in which the P-EII gene was highly expressed. The P-EII enzyme was inhibited by a serine protease inhibitor, diisopropyl fluorophosphate, as was the F-EII enzyme. Double reciprocal plots obtained from various concentrations of 6-aminohexanoate-dimer indicated that the kcat value of the P-EII enzyme (9.2 s-1) was approximately half that of the F-EII enzyme (19 s-1), and the P-EII enzyme had higher affinity toward this substrate (Km for P-EII, 0.6 mM; Km for F-EII, 15 mM). The P-EII enzyme had a temperature optimum of 48 degrees C, and a pH optimum of 7.5. It is speculated that since the P-nylB and F-nylB genes are more diverged from each other than the corresponding nylA genes, the latter may have evolved more recently.
Kanagawa
K
K
Department of Biotechnology, Osaka University, Japan.
Oishi
M
M
Negoro
S
S
Urabe
I
I
Okada
H
H
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D005075:Biological Evolution; D004269:DNA, Bacterial / Q000235:genetics; D004151:Dipeptides; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004151:Dipeptides; D000581:Amidohydrolases; C019683:6-aminohexanoate-cyclic-dimer hydrolase