Upward shift of the pH optimum of Acremonium ascorbate oxidase.
A gene encoding a thermostable Acremonium ascorbate oxidase (ASOM) was randomly mutated to generate mutant enzymes with altered pH optima. One of the mutants, which exhibited a significantly higher activity in the pH range 4.5-7 compared to ASOM, had a Gln183Arg substitution in the region corresponding to SBR1, one of the substrate binding regions of the zucchini enzyme. The other mutant with almost the same pH profile as Gln183Arg had a Thr527Ala substitution near the type 3 copper center and became more sensitive to azide than ASOM. Site-directed mutagenesis in the substrate binding regions with reference to the amino acid sequences of plant enzymes led to isolation of mutants shifted upward in the pH optimum; Val193Pro and Val193Pro/Pro190Ile increased the pH optimum by 1 and 0.5 units, respectively, while retaining the near-wild-type thermostability and azide sensitivity. The homology model of ASOM constructed from the zucchini enzyme coordinates suggested that replacement of Val193 by Pro could disturb the ion pair networks among Arg309, Glu192, Arg194 and Glu311. This perturbation could affect either the molecular recognition between the substrate and ASOM or the electron transfer from the substrate to the type 1 copper center, leading to the alkaline shift of the catalytic activity of the mutant enzyme. The other mutations, Val193Pro/Pro190Ile, could also induce similar structural perturbations involving the ion pair networks.
Sugino
Masayasu
M
Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Kajita
Sachiko
S
Banno
Koichi
K
Shirai
Tsuyoshi
T
Yamane
Takashi
T
Kato
Masashi
M
Kobayashi
Tetsuo
T
Tsukagoshi
Norihiro
N
D000164:Acremonium / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001204:Ascorbate Oxidase / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016415:Sequence Alignment
D005656:Fungal Proteins; D001204:Ascorbate Oxidase