Site-directed mutation at residues near the catalytic site of histamine dehydrogenase from Nocardioides simplex and its effects on substrate inhibition.
Histamine dehydrogenase from Nocardioides simplex (nHmDH) is a homodimer containing one 6-S-cysteinyl FMN (CFMN) and one [4Fe-4S] cluster per monomer. nHmDH catalyses the oxidative deamination of histamine to ammonia and imidazole acetaldehyde, but histamine inhibits its catalytic activity at high concentrations. We mutated gene-encoded residues (Tyr180, Gly268 and Asp269) near CFMN to understand the biophysical meaning of the substrate inhibition. Three mutants Y180F, G268D/D269C and Y180F/G268D/D269C were expressed by considering the DNA sequence alignment of histamine dehydrogenase from Rhizobium sp. 4-9 (rHmDH), which does not suffer from the substrate inhibition. The Y180F/G268D/D269C mutation to mimic rHmDH successfully suppressed the inhibition, although the catalytic activity decreased. The substrate inhibition was weakened by the Y180F mutation, but G268D/D269C was still susceptible to the inhibition. It was found that it also causes changes in the UV-vis absorption spectra of the substrate-reduced form and the redox potential of the enzymes. The characterization suggests that the thermodynamic preference of the semiquinone form of CFMN in the two-electron-reduced subunit of the enzyme is responsible for the substrate inhibition. However, destabilization of the semiquinone form leads to kinetic hindrance due to the uphill single electron transfer from the fully reduced CFMN to the [4Fe-4S] cluster.
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Maiko
M
Tsutsumi
Noriaki
N
Tsuse
Nobutaka
N
Fujieda
Kenji
K
Kano
D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009618:Nocardiaceae / Q000201:enzymology*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D007506:Iron-Sulfur Proteins; D000587:Oxidoreductases Acting on CH-NH Group Donors; C506665:histamine dehydrogenase