Cloning and characterization of histamine dehydrogenase from Nocardioides simplex.
Histamine dehydrogenase (NSHADH) can be isolated from cultures of Nocardioides simplex grown with histamine as the sole nitrogen source. A previous report suggested that NSHADH might contain the quinone cofactor tryptophan tryptophyl quinone (TTQ). Here, the hdh gene encoding NSHADH is cloned from the genomic DNA of N. simplex, and the isolated enzyme is subjected to a full spectroscopic characterization. Protein sequence alignment shows NSHADH to be related to trimethylamine dehydrogenase (TMADH: EC 1.5.99.7), where the latter contains a bacterial ferredoxin-type [4Fe-4S] cluster and 6-S-cysteinyl FMN cofactor. NSHADH has no sequence similarity to any TTQ containing amine dehydrogenases. NSHADH contains 3.6+/-0.3 mol Fe and 3.7+/-0.2 mol acid labile S per subunit. A comparison of the UV/vis spectra of NSHADH and TMADH shows significant similarity. The EPR spectrum of histamine reduced NSHADH also supports the presence of the flavin and [4Fe-4S] cofactors. Importantly, we show that NSHADH has a narrow substrate specificity, oxidizing only histamine (K(m)=31+/-11 microM, k(cat)/K(m)=2.1 (+/-0.4)x10(5)M(-1)s(-1)), agmatine (K(m)=37+/-6 microM, k(cat)/K(m)=6.0 (+/-0.6)x10(4)M(-1)s(-1)), and putrescine (K(m)=1280+/-240 microM, k(cat)/K(m)=1500+/-200 M(-1)s(-1)). A kinetic characterization of the oxidative deamination of histamine by NSHADH is presented that includes the pH dependence of k(cat)/K(m) (histamine) and the measurement of a substrate deuterium isotope effect, (D)(k(cat)/K(m) (histamine))=7.0+/-1.8 at pH 8.5. k(cat) is also pH dependent and has a reduced substrate deuterium isotope of (D)(k(cat))=1.3+/-0.2.
Limburg
Julian
J
Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA. jlimburg@ku.edu
Mure
Minae
M
Klinman
Judith P
JP
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005415:Flavins / Q000378:metabolism; D006632:Histamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D045563:Indolequinones / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009618:Nocardiaceae / Q000201:enzymology* / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013816:Thermodynamics; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism
D005415:Flavins; D045563:Indolequinones; D007506:Iron-Sulfur Proteins; C070569:tryptophan tryptophylquinone; D006632:Histamine; D014364:Tryptophan; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; C490533:histamine dehydrogenase, Nocardioides simplex