Systematic regulation of the enzymatic activity of phenylacetaldoxime dehydratase by exogenous ligands.
Phenylacetaldoxime dehydratase from Bacillus sp. OxB-1 (OxdB) contains a heme that acts as the active site for the dehydration reaction of aldoxime. Ferrous heme is the active form, in which the heme is five coordinate with His282 as a proximal ligand. In this work, we evaluated the functional role of the proximal ligand for the catalytic properties of the enzyme by "the cavity mutant technique". The H282G mutant of OxdB lost enzymatic activity, although the heme, which was five coordinate with a water molecule (or OH-) as an axial ligand, existed in the protein matrix. The enzymatic activity was rescued by imidazole or pyridine derivatives that acted as the exogenous proximal ligand. By changing the electron-donation ability of the exogenous ligand with different substituents, the enzymatic activity could be regulated systematically. The stronger the electron-donation ability of the exogenous ligand, the higher was the restored enzymatic activity. Interestingly, H282G OxdB with 2-methyl imidazole showed a higher activity than the wild-type enzyme. Kinetic analyses revealed that the proximal His regulated not only the affinity of substrate binding to the heme but also the elimination of the OH group from the substrate.
Kobayashi
Katsuaki
K
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
Kubo
Minoru
M
Yoshioka
Shiro
S
Kitagawa
Teizo
T
Kato
Yasuo
Y
Asano
Yasuhisa
Y
Aono
Shigetoshi
S
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004583:Electrons; D004789:Enzyme Activation / Q000235:genetics; D006420:Hemeproteins / Q000737:chemistry; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation; D010091:Oximes / Q000737:chemistry*; D013059:Spectrum Analysis, Raman
D001426:Bacterial Proteins; D006420:Hemeproteins; D007093:Imidazoles; D008024:Ligands; D010091:Oximes; C408881:phenylacetaldoxime; D008190:Lyases; C404242:oxd protein, Bacillus; C023728:acetaldehyde oxime