Functional analysis of molybdopterin biosynthesis in mycobacteria identifies a fused molybdopterin synthase in Mycobacterium tuberculosis

Abstract

Most mycobacterial species possess a full complement of genes for the biosynthesis of molybdenum cofactor (MoCo). However, a distinguishing feature of members of the Mycobacterium tuberculosis complex is their possession of multiple homologs associated with the first two steps of the MoCo biosynthetic pathway. A mutant of M. tuberculosis lacking the moaA1-moaD1 gene cluster and a derivative in which moaD2 was also deleted were significantly impaired for growth in media containing nitrate as a sole nitrogen source, indicating a reduced availability of MoCo to support the assimilatory function of the MoCo-dependent nitrate reductase, NarGHI. However, the double mutant displayed residual respiratory nitrate reductase activity, suggesting that it retains the capacity to produce MoCo. The M. tuberculosis moaD and moaE homologs were further analyzed by expressing these genes in mutant strains of M. smegmatis that lacked one or both of the sole molybdopterin (MPT) synthase-encoding genes, moaD2 and moaE2, and were unable to grow on nitrate, presumably as a result of the loss of MoCo-dependent nitrate assimilatory activity. Expression of M. tuberculosis moaD2 in the M. smegmatis moaD2 mutant and of M. tuberculosis moaE1 or moaE2 in the M. smegmatis moaE2 mutant restored nitrate assimilation, confirming the functionality of these genes in MPT synthesis. Expression of M. tuberculosis moaX also restored MoCo biosynthesis in M. smegmatis mutants lacking moaD2, moaE2, or both, thus identifying MoaX as a fused MPT synthase. By implicating multiple synthase-encoding homologs in MoCo biosynthesis, these results suggest that important cellular functions may be served by their expansion in M. tuberculosis.

FIG. 1.

Steps involved in the biosynthesis of bis-molybdopterin guanine dinucleotide cofactor from GTP. Annotations are as follows (from Tuberculist, http://tuberculist.epfl.ch): MoaA, MoCo biosynthesis protein A; MoaC, MoCo biosynthesis protein C; MoaD, MoCo biosynthesis protein D (MPT-converting factor small subunit); MoeB, MoCo biosynthesis protein (MPT synthase sulfurylase); MoaE, MoCo biosynthesis protein E (MPT-converting factor large subunit); Mog, MPT biosynthesis MOG protein; MoeA, MoCo biosynthesis protein MoeA; MobA, MGD biosynthesis protein A.

FIG. 2.

Genomic context of MoCo biosynthesis genes in M. tuberculosis H37Rv, M. bovis BCG Pasteur, and M. smegmatis mc²155. Genes conserved across all species are shown in red, those restricted to members of the MTBC are shown in black, and those unique to H37Rv are shown in gray.

FIG. 3.

Growth of M. tuberculosis strains on nitrate as a sole nitrogen source. The results are shown as the means and standard deviations from three experiments. Dashed lines indicate that the growth curve data are from the same experiment as shown in panel A.

FIG. 4.

HPLC profiles of MoCo form A standard and cell extracts from M. smegmatis strains. (A) MoCo form A standard (before dephosphorylation). (B) MoCo dephospho-form A standard. (C) Extract from M. smegmatis mc²155 spiked with a sample from panel B. (D) Extract from M. smegmatis mc²155. (E) Extract from M. smegmatis ΔmoaD2. (F) Extract from M. smegmatis ΔmoaD2(pMSmD2).

FIG. 5.

M. tuberculosis moaX has both moaD and moaE activities. The functions of mycobacterial moaD and moaE homologs in MoCo biosynthesis were analyzed by assessing the ability to complement the nitrate assimilation defect of M. smegmatis ΔmoaD2 and/or ΔmoaE2 mutants when ectopically expressed. Growth was assessed by OD₆₀₀. The data are shown as the means and standard deviations from three experiments.