Membrane-associated quinoprotein formaldehyde dehydrogenase from Methylococcus capsulatus Bath

Abstract

A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159-167, 1999; Vorholt et al., J. Bacteriol. 180:5351-5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b(559/569) complex as the physiological electron acceptor.

FIG. 1

Time course of metabolic fate profiles of [¹³C]formaldehyde in cell extracts from M. capsulatus Bath. ¹³C-NMR spectra of (A) a cell extract (34 mg of protein per ml) 10 min after the addition of [¹³C]formaldehyde and (B) ¹³C-NMR spectra of gaseous metabolites trapped from the headspace of the reaction vial in an insert that contained 6 M KOH. ¹³C-NMR spectra of (C) the washed membrane fraction (26 mg of protein per ml) 7 min after the addition of [¹³C]formaldehyde and (D) the same reaction mixture 14 min after the addition of [¹³C]formaldehyde. All peaks were normalized in reference to the internal standard (IS) dimethyl-d₆ sulfoxide and confirmed by authentic standards. Assay conditions are described in Materials and Methods. Abbreviations: 5,10-CH₂-THF, 5,10-methylenetetrahydrofolate; HCHO, formaldehyde; HMGSH, S-(hydroxymethyl)glutathione; HCOO⁻, formate; CO₂, carbon dioxide; and HCO₃⁻, bicarbonate.

FIG. 2

SDS-polyacrylamide slab gel electrophoresis of fractions during the purification of DL-FalDH by method I from M. capsulatus Bath. Washed membrane fraction (lane A), detergent extract (lane B), post-DEAE-Sepharose FF (lane C), post-phenyl-Sepharose (lane D), post-Resource Q (lane E), and low-range SDS Bio-Rad protein standards (lane F).

FIG. 3

Purification and characterization of prosthetic group from DL-FalDH by HPLC ES-MS. The evaporative light-scattering profile of the prosthetic group following conversion of the ketal into putative PQQ through the hexoxy-PQ intermediate. The insets show the diode array wavelength spectrum (λ_max = 205, 248, and 332 nm) and positive-ion electrospray mass spectrum ([M + H] + = 331.2 m/z) for the major species (>83% purity) of the conversion reaction that eluted at 8.7 min.

FIG. 4

N-terminal amino acid sequence of DL-FalDH from M. capsulatus (Mc) Bath, the sulfide quinone reductase from Oscillatoria limnetica (Ol) (4), and the sulfide quinone reductase from Rhodobacter capsulatus (Rc) (39). The fingerprint of the βαβ fold of the NAD(P)/FAD binding domain (16, 19, 52) is also shown. Amino acid residues matching the fingerprint of the βαβ fold of the NAD(P)/FAD binding domain are in boldface type.

FIG. 5

Proposed pathways of methane oxidation in M. capsulatus Bath. Membrane-associated proteins are shown above and soluble proteins below the substrate oxidation steps. With the exception of the methane oxidation by the pMMO, complex I, and the cytochrome bc₁ complex, the enzymes and physiological electron acceptors/donors involved in the oxidation of methane to carbon dioxide have been characterized in a number of methanotrophs (1, 3, 22, 34, 44, 46, 51, 54). Abbreviations: complex I, NADH: quinone oxidoreductase; cyt., cytochrome; MDH, methanol dehydrogenase; NAD(P)-FalDH, NAD- and NADP-linked formaldehyde dehydrogenases; FDH, formate dehydrogenase.