Aromatic amine dehydrogenase, a second tryptophan tryptophylquinone enzyme

Abstract

Aromatic amine dehydrogenase (AADH) catalyzes the oxidative deamination of aromatic amines including tyramine and dopamine. AADH is structurally similar to methylamine dehydrogenase (MADH) and possesses the same tryptophan tryptophylquinone (TTQ) prosthetic group. AADH exhibits an alpha 2 beta 2 structure with subunit molecular weights of 39,000 and 18,000 and with a quinone covalently attached to each beta subunit. Neither subunit cross-reacted immunologically with antibodies to the corresponding subunits of MADH, and the N-terminal amino acid sequence of the beta subunit of AADH exhibited no homology with the highly conserved beta subunits of MADH. The absorption spectra for the oxidized, semiquinone, and reduced forms of AADH have been characterized, and extinction coefficients for the absorption maxima of each redox form have been determined. These spectra are very similar to those for MADH, indicating the likelihood of a TTQ cofactor. This was verified by the near identity of the vibrational frequencies and intensities in the resonance Raman spectra for the oxidized forms of AADH and MADH. A stable semiquinone of AADH could be observed during a reductive titration with dithionite, whereas titration with tyramine proceeded directly from the oxidized to the reduced form. AADH was very stable against denaturation by heat and exposure to guanidine. The individual subunits could be separated by gel filtration after incubation in guanidine hydrochloride, and partial reconstitution of activity was observed on recombination of the subunits. Steady-state kinetic analysis of AADH yielded a Vmax of 17 mumol/min/mg and a Km for tyramine of 5.4 microM. Substrate inhibition by tyramine was observed. AADH was irreversibly inhibited by hydrazine, phenylhydrazine, hydroxylamine, semicarbazide, and aminoguanidine. Isonicotinic acid hydrazide (isoniazid) and isonicotinic acid 2-isopropyl hydrazide (iproniazid) were reversible noncompetitive inhibitors of AADH and exhibited K(i) values of 8 and 186 microM, respectively. The similarities and differences between AADH and other amine oxidizing enzymes are also discussed.

Cloning, sequencing and mutagenesis of the genes for aromatic amine dehydrogenase from Alcaligenes faecalis and evolution of amine dehydrogenases The GenBank accession number for the aau gene cluster from Alcaligenes faecalis is AF302652.

“…This fragment was completely sequenced and three open reading frames (ORFs) were identified in it. One of these ORFs was truncated and encoded a polypeptide containing the previously determined N-terminal sequence of the AADH small subunit polypeptide (Govindaraj et al, 1994). Two other ORFs encoded polypeptides highly homologous to MauE and MauD and were called aauE and aauD.…”

“…The general strategy of construction and analysis of partial gene libraries was as described by us earlier (Chistoserdov et al, , 1994b. The oligonucleotide AC14 was designed based on the N-terminal sequence of the AADH small subunit polypeptide (GADHII ; Govindaraj et al, 1994). A partial clone library was constructed in the vector pRK310, using the fraction of the BclI-BglII digest of the A. faecalis chromosome, which hybridized to AC14 (not shown).…”

“…AADH has been found so far only in Alcaligenes faecalis during growth of the bacterium on medium containing phenylethylamine (Nozaki, 1987). AADH resembles MADH in many of its properties (Govindaraj et al, 1994). It has two large and two small subunits with molecular masses of 39 and 18 kDa, respectively.…”

Distribution of amine oxidases and amine dehydrogenases in bacteria grown on primary amines and characterization of the amine oxidase from Klebsiella oxytoca

“…pneumoniae) (Yamashita et af., 1993). Surprisingly, other Gram-negative bacteria convert these substrates via quite different types of oxidoreductases : Alcaligenes faecafis with a periplasmic, TTQ-containing amine dehydrogenase (Govindaraj et al, 1994) ; Pseudomonas aeruginosa with a membranebound, uncharacterized amine dehydrogenase (Cuskey et af., 1987) ; Pseudomonas putida with a soluble haemcontaining amine dehydrogenase (Durham & Perry, 1978); Sarcina Zutea with a flavoprotein amine oxidase (Kumagai et af., 1969). At present, the reason for this diversity of enzymes catalysing the same reaction, and having similar substrate specificity, is unknown.…”

New pathway of amine oxidation respiratory chain of Paracoccus denitrificans IFO 12442

“…These substrates are oxidized to the corresponding aldehydes by specific amine dehydrogenases, which function in the periplasmic space of the Gram-negative bacteria. Much effort has been devoted to understanding the structure and function of methylamine dehydrogenase (MADH) [1±5] and aromatic amine dehydrogenase (AADH) [6,7]. Their prosthetic group is known to be tryptophan tryptophylquinone (TTQ).…”