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.

Production of completely flavinylated histamine dehydrogenase, unique covalently bound flavin, and iron-sulfur cluster-containing enzyme of nocardioides simplex in Escherichia coli, and its properties.

The hmd gene of histamine dehydrogenase from Nocardioides simplex was overexpressed in Escherichia coli, and the resulting enzyme was purified to homogeneity. The purified recombinant enzyme is almost identical with the native enzyme in view of molecular weight and specific activity, and is stoichiometrically assembled with the three cofactors 6-S-cysteinyl FMN, 4Fe-4S cluster, and ADP.

6-S-cysteinyl flavin mononucleotide-containing histamine dehydrogenase from Nocardioides simplex: molecular cloning, sequencing, overexpression, and characterization of redox centers of enzyme.

Histamine dehydrogenase from Nocardioides simplex is a homodimeric enzyme and catalyzes oxidative deamination of histamine. The gene encoding this enzyme has been sequenced and cloned by polymerase chain reactions and overexpressed in Escherichia coli. The sequence of the complete open reading frame, 2073 bp coding for a protein of 690 amino acids, was determined on both strands. The amino acid sequence of histamine dehydrogenase is closely related to those of trimethylamine dehydrogenase and dimethylamine dehydrogenase containing an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-flavin mononucleotide, and one 4Fe-4S cluster as redox active cofactors in each subunit of the homodimer. The presence of the identical redox cofactors in histamine dehydrogenase has been confirmed by sequence alignment analysis, mass spectral analysis, UV-vis and EPR spectroscopy, and chemical analysis of iron and acid-labile sulfur. These results suggest that the structure of histamine dehydrogenase in the vicinity of the two redox centers is almost identical to that of trimethylamine dehydrogenase as a whole. The structure modeling study, however, demonstrated that a putative substrate-binding cavity in histamine dehydrogenase is quite distinct from that of trimethylamine dehydrogenase.