Affiliation:
1. Department of Microbiology1 and
2. Department of Biochemistry,2 National University of Singapore, 119260 Singapore
Abstract
ABSTRACT
Two 3-hydroxybenzoate-inducible gentisate 1,2-dioxygenases were purified to homogeneity from
Pseudomonas alcaligenes
NCIB 9867 (P25X) and
Pseudomonas putida
NCIB 9869 (P35X), respectively. The estimated molecular mass of the purified P25X gentisate 1,2-dioxygenase was 154 kDa, with a subunit mass of 39 kDa. Its structure is deduced to be a tetramer. The pI of this enzyme was established to be 4.8 to 5.0. The subunit mass of P35X gentisate 1,2-dioxygenase was 41 kDa, and this enzyme was deduced to exist as a dimer, with a native molecular mass of about 82 kDa. The pI of P35X gentisate 1,2-dioxygenase was around 4.6 to 4.8. Both of the gentisate 1,2-dioxygenases exhibited typical saturation kinetics and had apparent
K
m
s of 92 and 143 μM for gentisate, respectively. Broad substrate specificities were exhibited towards alkyl and halogenated gentisate analogs. Both enzymes had similar kinetic turnover characteristics for gentisate, with
k
cat
/
K
m
values of 44.08 × 10
4
s
−1
M
−1
for the P25X enzyme and 39.34 × 10
4
s
−1
M
−1
for the P35X enzyme. Higher
k
cat
/
K
m
values were expressed by both enzymes against the substituted gentisates. Significant differences were observed between the N-terminal sequences of the first 23 amino acid residues of the P25X and P35X gentisate 1,2-dioxygenases. The P25X gentisate 1,2-dioxygenase was stable between pH 5.0 and 7.5, with the optimal pH around 8.0. The P35X enzyme showed a pH stability range between 7.0 and 9.0, and the optimum pH was also 8.0. The optimal temperature for both P25X and P35X gentisate 1,2-dioxygenases was around 50°C, but the P35X enzyme was more heat stable than that from P25X. Both enzymes were strongly stimulated by 0.1 mM Fe
2+
but were completely inhibited by the presence of 5 mM Cu
2+
. Partial inhibition of both enzymes was also observed with 5 mM Mn
2+
, Zn
2+
, and EDTA.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology