Characterization of two methanopterin biosynthesis mutants of Methylobacterium extorquens AM1 by use of a tetrahydromethanopterin bioassay

Abstract

An enzymatic assay was developed to measure tetrahydromethanopterin (H(4)MPT) levels in wild-type and mutant cells of Methylobacterium extorquens AM1. H(4)MPT was detectable in wild-type cells but not in strains with a mutation of either the orf4 or the dmrA gene, suggesting a role for these two genes in H(4)MPT biosynthesis. The protein encoded by orf4 catalyzed the reaction of ribofuranosylaminobenzene 5'-phosphate synthase, the first committed step of H(4)MPT biosynthesis. These results provide the first biochemical evidence for H(4)MPT biosynthesis genes in bacteria.

FIG. 1.

Reaction of MtdB. The R group represents the side chain of H₄MPT, which consists of ribitol, ribofuranosyl phosphate, and hydroxglutaryl groups. H₄SPT from Methanosarcina thermophila contains an additional glutamate residue, while dephospho-H₄MPT from M. extorquens lacks the phosphate and hydroxyglutaryl groups of H₄MPT. Formaldehyde addition can occur nonenzymatically; however, in cells of M. extorquens, the reaction is catalyzed enzymatically by the formaldehyde-activating enzyme (31).

FIG. 2.

Detection of H₄MPT in methanogen cell extracts by the MtdB assay. The complete reaction mixture (line 1) contained 120 mM KH₂PO₄ (pH 6.8), 3 mM formaldehyde, 20 μl of Ni-NTA-purified His₆-MtdB (15 μg of protein), and 100 μl of Methanosarcina thermophila heat-treated cell extract. After a stable baseline was established at 340 nm for 25 s, the reaction was initiated with 100 μl of 2 mM NAD⁺. Control assays contained all of the components except His₆-MtdB (line 2), NAD⁺ (line 3), heated cell extract (line 4), or formaldehyde (line 5).

FIG. 3.

Detection of dephospho-H₄MPT in extracts of M. extorquens AM1. Cell extracts were prepared and concentrated as described in the text. The assay components were the same as those described in the legend to Fig. 2. Cell extracts were from wild-type AM1 grown on 0.5% methanol (line 1), wild-type AM-1 grown on 20 mM succinate (line 2), fae mutant cells grown on succinate (line 3), orf4 mutant cells grown on succinate (line 4), or dmrA mutant cells grown on succinate (line 5).