Microbial oxidation of amines. Spectral and kinetic properties of the primary amine dehydrogenase of Pseudomonas AM 1

Abstract

1. An improved procedure is reported for purification of the amine dehydrogenase from methylamine-grown Pseudomonas AM1 which yielded a product homogeneous by sedimentation and disc-electrophoretic analysis, with molecular weight of 133000. 2. The purified enzyme had absorption maxima at 280 and 430nm. On aging, a third peak appeared at 325nm, and the 430nm peak decreased in intensity. This spectrum was independent of pH. 3. Addition of 2.5mm-semicarbazide, phenylhydrazine, hydrazine or hydroxylamine produced modified spectra with maxima respectively at 400, 440, 395 and 425nm. 4. Aerobic addition of methylamine resulted in a bleaching of the 430nm peak and the appearance of a new one at 325nm. This spectral change was retained after removal of the methylamine by dialysis. The original spectrum could be restored on addition of phenazine methosulphate. 5. Addition of borohydride partially inactivated the enzyme and produced spectral changes similar to those observed with methylamine. Pre-treatment with methylamine prevented the inactivation by borohydride. The degree of inactivation could be increased by alternate phenazine methosulphate and borohydride treatments. 6. The addition of methylamine or borohydride each caused shifts in the fluorescence emission maximum from 348 to 380nm. 7. Lineweaver-Burk plots of reciprocal activity against reciprocal concentration of either of the substrates n-butylamine or phenazine methosulphate were consistent with a mechanism that involves interconversion of two free forms of the enzyme by the two substrates. 8. The enzyme, although spectrally modified, was not inactivated by dialysis against diethyldithiocarbamate, and contained about 0.27 g-atom of copper/mol, with small traces of cobalt, iron and zinc. 9. Conventional methods of resolution did not release the prosthetic group. Heat denaturation after treatment of the enzyme with methylamine liberated a yellow chromophore which did not reactivate resolved aspartate aminotransferase, and whose spectral, electrophoretic and fluorescence properties did not agree with any recognizable pyridoxal derivatives. 10. Despite the inconclusive results with the isolated chromophore, the observations on the enzyme suggest that it may contain a pyridoxal derivative bound as a Schiff's base which is converted into the pyridoxamine form on aerobic treatment with methylamine and reconverted into the pyridoxal form with phenazine methosulphate. 11. The copper detected is probably not involved in the enzyme mechanism, since most copper-chelating agents are not inhibitory, and since the enzyme does not react with oxygen.

Mutational Analysis of Mau Genes Involved in Methylamine Metabolism in Paracoccus Denitrificans

“…The 285-nm peak originates from fluorescing tryptophan residues in the protein and the 328-nm peak from the reduced form of TTQ ('ITQH,) in MADH. These findings are in agreement with those reported for the isolated MADH from M. extorquens AM1 (Eady and Large, 1971). Methylamine-induced fluorescence of these cell-free extracts thus appears to reflect the presence of an active MADH capable of methylamine oxidation.…”

Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1

“…These groups include facultative autotrophs such as Paracoccus denitrificans (25) and Thiobacillus versutus A2 (23); facultative methylotrophs such as pink-pigmented Methylobacterium (formerly "Pseudomonas") spp. (6,10,18,19,37), Hyphomicrobium spp. (36,45,56), and nonpigmented "Pseudomonas" spp.…”

A new model for the pro‐PQQ cofactor of quinoprotein methylamine dehydrogenase

“…The model proposed in the current paper for the pro-PQQ cofactor of MADH also contains such a peptide bond, linking the Cy (or C9 in the notation for the PQQ cofactor) carboxylate group of the quinone indole bound Glu-57 to the Ne atom of Arg-107. Since the reactions catalyzed by these enzymes differ only at the level of the transfer of electrons from the substrate to the respective electron acceptors, and since their respective cofactors are known to be similar [4], the diamine oxidase enzymes might also contain such an 'open-form' pro-PQQ cofactor.…”

“…With respect to the 'covalent' class of quinoproteins, it is interesting to consider the enzyme diamine oxidase, whose cofactor bears resemblance to that of MADH [4]. Recently, results were reported of the sequence analysis of a tryptic peptide containing the phenylhydrazone adduct of the cofactor, obtained from the pig kidney enzyme [20].…”

“…In the early stage of the characterization of this enzyme, it became obvious that it contained a very tightly bound organic cofactor, which resembled that of the copper-containing diamine oxidase enzymes (EC 1. 4.3.6) [4]. Later, ESR and ENDOR spectra of the native enzyme indicated that the cofactor had a quinone structure, and that it was very Since the latter was identified as pyrroloquinoline quinone (or PQQ) [6,7], the MADH cofactor was also assumed to be PQQ, or a closely related compound.…”