Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans

Abstract

Methylamine dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methylamine-grown cells. The enzyme exhibited a pI value of 4.3 and was composed of two 46,700-dalton subunits and two 15,500-dalton subunits. Each small subunit possessed a covalently bound pyrrolo-quinoline quinone prosthetic group. The amino acid compositions of the large and small subunits are very similar to those of other methylamine dehydrogenases which have been isolated from taxonomically different sources. It has been established recently that certain oxidoreductases from a variety of sources contain pyrrolo-quinoline quinone (PQQ) as a prosthetic group (2, 4, 10, 11). These quinoproteins include bacterial methanol (9) and glucose dehydrogenases (12), which possess noncovalently associated PQQ, and bacterial methylamine dehydrogenase, which contains a covalently bound form of PQQ (8,21). Mammalian plasma amine oxidase (26) and choline dehydrogenase (3) also possess covalently attached PQQ. When grown on methylamine as a sole source of carbon and energy, Paracoccus denitrificans synthesizes a methylamine dehydrogenase which functions in the periplasm of this gramnegative bacterium and donates electrons to a periplasmic type I blue copper protein, amicyanin (16). We have previously reported the partial purification of methylamine dehydrogenase from P. denitrificans (16) and have characterized the physical and redox properties of amicyanin (14,16,18,25) and periplasmic c-type cytochromes (14, 17) which accept electrons from methylamine dehydrogenase via amicyanin. This paper reports a relatively simple procedure by which methylamine dehydrogenase was purified to homogeneity from P. denitrificans and describes several of the physical and kinetic properties of this enzyme. MATERIALS AND METHODSBacterial strains and culture conditions. P. denitrificans (ATCC 13543) was grown aerobically at 30°C in the medium of Kornberg and Morris (23), supplemented with 0.05% NaHCO3, 0.01% yeast extract, and 0.5% methylamine, 0.5% methanol, or 0.3% succinate.Preparation of proteins. Methylamine dehydrogenase was purified from the periplasmic fraction of methylamine-grown cells, which was prepared by the method of Alefounder and Ferguson (1). Routinely, 15 to 20 g (wet weight) of cells was * Corresponding author. fractionated at a time, and four such preparations were pooled, concentrated by ultrafiltration over an Amicon YM5 membrane (Amicon Corp., Lexington, Mass.), dialyzed against 20 mM potassium phosphate (pH 7.2), and applied to a DEAE-cellulose column (3.5 by 30 cm) which had been equilibrated in the same buffer that was used for dialysis. This column was eluted with a linear gradient (2.0 liters) of 0 to 400 mM NaCl in the starting buffer. Fractions containing methylamine dehydrogenase were pooled, concentrated by ultrafiltration over an Amicon PM 30 membrane, dialyzed against 20 mM potassium phosphate (pH 7.2), and applied to a DEAE-Trisacryl (LKB Instruments, Inc., Rockville, Md.) column...

Mutants of Methylobacterium extorquens and Paracoccus denitrificans deficient in c‐type cytochrome biogenesis synthesise the methylamine‐dehydrogenase polypeptides but cannot assemble the tryptophan‐tryptophylquinone group

“…The only previously reported condition for the induction of methylamine dehydrogenase in P. denitrifcans was growth on this carbon source (Husain and Davidson, 1987). Neither methanol nor methylamine dehydrogenases were expressed following growth of R denitrificans with succinate in the presence of methylamine (Table 1).…”

“…Methylamine dehydrogenase has a periplasmic location in l? denitrificans (Husain and Davidson, 1987). While this has not been demonstrated directly in M. extorquens [which does not release periplasmic proteins when either incubated with lysozyme or subjected to osmotic shock (Anthony, C., unpublished results), sequencing of the structural genes for the H and L subunits of the enzyme in this organism reveal that both proteins are synthesised with N-terminal signal peptides (Chistoserdov et al, 1990;Chistoserdov and Lidstrom, 1991 ;Lidstrom and Chistoserdov, 1993), indicating a periplasmic location for the mature enzyme.…”

“…Paracoccus denitrificans) (Eady and Large, 1968;Haywood et al, 1982;Husain and Davidson, 1987). The enzymes have an a& subunit structure with a heavy (H) subunit of 40-45 kDa and light (L) subunit of 14-15 kDa; in all known cases the enzyme is periplasmic (Burton et al, 1983;Husain and Davidson, 1987;Ubbink et al, 1991;Chistoserdov et al, 1992). The methylamine dehydrogenase prosthetic group, recently shown to be a unique quinonoid moiety termed tryptophan tryptophylquinone (TTQ), is formed from two tryptophan residues (positions 55 and 106 in the M .…”

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.

“…Until recently, methylamine dehydrogenase (MADH) was the only known amine dehydrogenase. MADH enzymes from different sources consist of two small and two large subunits with molecular masses ranging from 13 to 15 kDa and from 40 to 45 kDa, respectively, and in all known cases this enzyme is periplasmic (Eady & Large, 1968 ;Matsumoto, 1978 ;Haywood et al, 1982 ;Kenny & McIntire, 1983 ;Husain & Davidson, 1987 ;Kiriuchin et al, 1990). The small subunit has a quinone prosthetic group called tryptophan tryptophylquinone (TTQ), which is synthesized from two tryptophans of the MADH small subunit polypeptide (McIntire et al, 1991).…”

Organization of methylamine utilization genes (mau) in ‘Methylobacillus flagellatum’ KT and analysis of mau mutants

“…These bacteria include the facultative autotrophs Paracoccus denitrificans (Husain & Davidson, 1987) and Thiobacillus uersutus (Haywood et at., 1982), the facultative methylotroph Methylobacterium extorquens AM1 (Eady & Large, 1968) belonging to the 01 subgroup of proteobacteria, the restricted facultative methylotroph Methylophilus methylotrophus W3A1 (Kenny & Mclntire, 1983) and the obligate methylotrophs ' Methylobacillus flagellaturn ' KT (Kiriukhin et al, 1990), Methylomonas sp. J.…”