Methylamine oxidase from Arthrobacter P1. A bacterial copper-quinoprotein amine oxidase

Abstract

Methylamine oxidase from Arthrobacter P1 was purified to homogeneity. The enzyme oxidizes primary amines but not tyramine or polyamines like spermine and putrescine. The enzyme activity has a pH optimum of 8.0 with methylamine, and is inhibited by certain cations as well as anions at rather low concentrations. The enzyme has an M , of 167900, an isoelectric point of 4.6, consists of two (probably identical) subunits (Mr 82250) and contains two copper atoms but no sugar residues. The visible absorption spectra of the enzyme as it is isolated (broad maximum at 480 nm), that of its reduced form obtained on addition of excess of methylamine (maximum at 470 nm) and that of phenylhydrazine-inhibited enzyme (maximum at 440 nm) are very similar to those of eucaryotic copper-containing amine oxidases (EC 1.4.3.6). Also the stoichiometry of inhibition with carbonyl group reagents is similar since the enzyme reacted with only one methylhydrazine. The adduct isolated from copper-free enzyme, treated with 2,4-dinitrophenylhydrazine, was identical to that found in bovine serum amine oxidase treated with t h s compound after copper removal. This indicates that the enzyme is a copper-quinoprotein amine oxidase, the first example from bacterial origin.A large number of methylotrophic bacteria are able to convert methylamine directly into formaldehyde and ammonia [I]. Within this group, all gram-negative bacteria studied so far use NAD(P)-independent, periplasma-located methylamine dehydrogenase (EC 1.4.99.3) for that purpose. [5], and probably also from yeasts [6], are copper-containing enzymes. In this light it was interesting to purify methylamine oxidase from Arthrobacter P1 and to look for the presence of flavin and copper.During these studies, it was found that this enzyme had all the characteristics of a copper-containing amine oxidase. Since such an enzyme also contains an organic prosthetic group, the nature of which was recently established to be pyrroloquinoline quinone (PQQ) in bovine serum amine oxidase [7], attempts were made to provide evidence for the existence of this compound in methylamine oxidase. MATERIALS AND METHODS Cell culturingArthrobacter P1 (kindly provided by Prof. W. Harder) was cultured aerobically at 30°C on a mineral medium 121 with 0.5% methylamine . HCl as a carbon and energy source. The cells were harvested in the stationary phase, washed with 50 mM potassium phosphate, pH 7.0, and stored at -20°C. Enzyme purificationThawed cells (84 g wet weight) were suspended in 125 ml 50 mM potassium phosphate, pH 7.0, and treated during 5 min by an ultrasonic disintegrator (MSE, 150 W) with cooling. After centrifugation, the pellet was resuspended in 100 ml buffer and the sonification repeated for 10 min. The supernatants were combined (225 ml) and 24.3 g poly(ethy1-ene glycol) (PEG) 6000 was added (10% w/v) with stirring for 20 min at room temperature. After centrifugation, further protein precipitation was achieved by adding 155.7 g PEG 6000 to the supernatant (50% w/v) and stirring for 90 min. Precipi...

Coordination chemistry of copper-containing amine oxidases: nuclear magnetic relaxation dispersion studies of copper binding, solvent-water exchange, substrate and inhibitor binding, and protein aggregation

“…Azide perturbs the NMRD of PPAO to a greater extent than that of the other amine oxidases examined, as indicated in Table I. This result is consistent with a previous report, which suggested that, at 25 °C, azide could displace at most approximately 50% of the coordinated water that contributed to solvent relaxation via rapid exchange.27 Our NMRD measurements were made at 5 °C, where previous results indicate that the binding of anions [Protein] = 0.05 mM in 50 mM KP04 (pH 7.0). Note: The actual copper content of the apo sample is 3% (0.06:1 = Cu(II):proteln).…”

Cloning of the maoA Gene that Encodes Aromatic Amine Oxidase of Escherichia coli W3350 and Characterization of the Overexpressed Enzyme

“…The first bacterial one was methylamine oxidase from the grampositive bacterium Arthrobacter PI (van Iersel et al, 1986). Aromatic and aliphatic amine oxidases, probably of the quinoprotein type, have been detected in yeasts, and aromatic and aliphatic enzymes have been found in the same strains (Haywood and Large, 1981 ;Bruinenberg et al, 1989;Mu et al, 1992).…”

Methylamine Utilization via the N -Methylglutamate Pathway in Methylobacterium extorquens PA1 Involves a Novel Flow of Carbon through C 1 Assimilation and Dissimilation Pathways

“…In many Gram-negative methylotrophs, such as Methylobacterium extorquens AM1, MA is oxidized by a periplasmic methylamine dehydrogenase (MaDH) (8,9) to formaldehyde. Methylamine oxidase, a Cu-containing enzyme present in Gram-positive methylotrophs (10) and methylotrophic yeast strains (11), oxidizes MA to formaldehyde (12) in a single step as well. In contrast, the N-methylglutamate (NMG) pathway, originally described in Pseudomonas spp.…”