Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol

Abstract

Formaldehyde is toxic for all organisms from bacteria to humans due to its reactivity with biological macromolecules. Organisms that grow aerobically on single-carbon compounds such as methanol and methane face a special challenge in this regard because formaldehyde is a central metabolic intermediate during methylotrophic growth. In the alpha-proteobacterium Methylobacterium extorquens AM1, we found a previously unknown enzyme that efficiently catalyzes the removal of formaldehyde: it catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin, a reaction which also proceeds spontaneously, but at a lower rate than that of the enzyme-catalyzed reaction. Formaldehyde-activating enzyme (Fae) was purified from M. extorquens AM1 and found to be one of the major proteins in the cytoplasm. The encoding gene is located within a cluster of genes for enzymes involved in the further oxidation of methylene tetrahydromethanopterin to CO(2). Mutants of M. extorquens AM1 defective in Fae were able to grow on succinate but not on methanol and were much more sensitive toward methanol and formaldehyde. Uncharacterized orthologs to this enzyme are predicted to be encoded by uncharacterized genes from archaea, indicating that this type of enzyme occurs outside the methylotrophic bacteria.

FIG. 1

C₁ metabolism of M. extorquens AM1. Formaldehyde is produced in the periplasm of the cell from methanol by a pyrroloquinoline quinone-dependent methanol dehydrogenase and crosses the membrane. Cytoplasmic formaldehyde reacts with either H₄F or H₄MPT. The condensation of formaldehyde and H₄MPT is catalyzed by Fae (this study). The C₁ unit is utilized for further oxidation to CO₂ or incorporated via the serine cycle. a₁, NADP⁺-dependent methylene H₄F dehydrogenase MtdA (33); a₂, methenyl H₄F cyclohydrolase FchA (27); a₃, formyl H₄F synthase; a₄, formate dehydrogenase; b₁, NAD(P)⁺-methylene H₄MPT dehydrogenases MtdA and MtdB (12, 33); b₂, methenyl H₄MPT cyclohydrolase Mch (27); b₃, formyl methanofuran:H₄MPT formyltransferase; b₄, formyl methanofuran dehydrogenase.

FIG. 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of fractions of Fae upon purification from M. extorquens AM1. Lane M, molecular mass standards (Amersham-Pharmacia Biotech); lane 1, 20 μg of cell extract protein; lane 2, 8 μg of protein after DEAE-Sephacel; lane 3, 6 μg of protein after Q-Sepharose; lane 4, 4 μg of protein after hydroxyapatite.

FIG. 3

pH optimum of the spontaneous reaction of formaldehyde with H₄MPT and of the reaction catalyzed by Fae. The assays contained 120 mM potassium phosphate, 40 μM H₄MPT, 1.6 mM CH₂O, and 5 μg of purified Fae when indicated. Assays were performed at the pH value indicated at 30°C. ●, activity in the absence of enzyme (spontaneous rate); ▴, activity in the presence of enzyme; ▾, activity in the presence of enzyme corrected for the spontaneous rate.

FIG. 4

Kinetics of Fae with respect to different formaldehyde and H₄MPT concentrations. The assay mixtures contained 50 mM Tricine-KOH (pH 8.0), 30 mM MgCl₂, and 1.5 μg of purified Fae; the H₄MPT concentration was 50 μM (A) or as indicated (B), and the CH₂O concentration was as indicated (A) or 1.6 mM (B).

FIG. 5

(A) Alignment of the amino acid sequence of Fae from M. extorquens AM1 and putative proteins from the complete genomes of the sulfate-reducing archaeon A. fulgidus (AF1305) (21) and the methanogenic archaeon M. thermoautotrophicum ΔH (Mth1474) (31). (B) Sequence analysis of orthologs of Fae from M. extorquens AM1. Fae from M. extorquens AM1 shows sequence identity to the N-terminal domain of putative proteins from A. fulgidus, M. thermoautotrophicum ΔH, and M. jannaschii (4, 21, 31). The C-terminal domains of these archaeal proteins themselves show sequence identity to 3-hexulose-6-phosphate synthase (HPS; RmpA) from M. aminofaciens (35). A homologue of RmpA is linked to the 6-phospho-4-hexuloisomerase (PHI; RmpB) homologue from M. aminofaciens (29) in Pyrococcus species (19).