Unexpected abundance of coenzyme F(420)-dependent enzymes in Mycobacterium tuberculosis and other actinobacteria

Abstract

Regimens targeting Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), require long courses of treatment and a combination of three or more drugs. An increase in drug-resistant strains of M. tuberculosis demonstrates the need for additional TB-specific drugs. A notable feature of M. tuberculosis is coenzyme F(420), which is distributed sporadically and sparsely among prokaryotes. This distribution allows for comparative genomics-based investigations. Phylogenetic profiling (comparison of differential gene content) based on F(420) biosynthesis nominated many actinobacterial proteins as candidate F(420)-dependent enzymes. Three such families dominated the results: the luciferase-like monooxygenase (LLM), pyridoxamine 5'-phosphate oxidase (PPOX), and deazaflavin-dependent nitroreductase (DDN) families. The DDN family was determined to be limited to F(420)-producing species. The LLM and PPOX families were observed in F(420)-producing species as well as species lacking F(420) but were particularly numerous in many actinobacterial species, including M. tuberculosis. Partitioning the LLM and PPOX families based on an organism's ability to make F(420) allowed the application of the SIMBAL (sites inferred by metabolic background assertion labeling) profiling method to identify F(420)-correlated subsequences. These regions were found to correspond to flavonoid cofactor binding sites. Significantly, these results showed that M. tuberculosis carries at least 28 separate F(420)-dependent enzymes, most of unknown function, and a paucity of flavin mononucleotide (FMN)-dependent proteins in these families. While prevalent in mycobacteria, markers of F(420) biosynthesis appeared to be absent from the normal human gut flora. These findings suggest that M. tuberculosis relies heavily on coenzyme F(420) for its redox reactions. This dependence and the cofactor's rarity may make F(420)-related proteins promising drug targets.

FIG. 1.

Flavonoid cofactor structures. (A) FMN. (B) Coenzyme F₄₂₀. Note that coenzyme F₄₂₀ typically contains 5 to 7 side chain glutamate residues in mycobacterial species (3).

FIG. 2.

Average numbers of putative F₄₂₀/FMN-binding protein family genes in actinobacterial species. The presence of F₄₂₀ biosynthesis components is correlated with large expansions of these families.

FIG. 3.

SIMBAL analysis of the M. tuberculosis FGD1 gene (Rv0407) versus a partition of the LLM family (PF00296) based on the ability (positive branch) of source genomes to produce cofactor F₄₂₀. Closely related homologs of the TIGR03557 family were removed from the positive training set to accentuate features common to all F₄₂₀-dependent LLM family sequences. (Top left) Raw SIMBAL data (log likelihood scores). (Top right) Normalized data are represented as SIMBAL scores divided by the sequence window length in order to identify prominent localized regions (colored circles). (Bottom) Sequence of Rv0407. High-scoring subsequence regions are indicated in colors corresponding to the circles in the top right panel. Underlined residues make contacts (≤3.5 Å) with the F₄₂₀ cofactor in the crystal structure under Protein Data Bank (PDB) accession no. 3B4Y (4), starred residues make contacts with the citrate molecule bound in the putative substrate cavity, and dotted residues contribute to a positively charged patch in an extended surface cleft adjacent to the end of the resolved part of the polyglutamate cofactor side chain (see Fig. 4B).

FIG. 4.

(A) SIMBAL-identified residues making up the F₄₂₀-binding surface of M. tuberculosis FGD1 (PDB accession no. 3B4Y [4]). Peak 1, SDH, is in contact with the carboxylate oxygens of the deazaflavin terminal ring (cyan). Peak 2, SVLT, includes the nonproline cis-peptide bond between serine and valine (2) and comprises the “bulge” behind the deazaflavin central ring (red). Peak 3, GTGE, is in contact with the phospholactate component of the side chain (yellow). Peak 4, FKER, is in contact with the single glutamate resolved by the crystal structure and forms a long adjacent surface cleft (blue). Peak 5, AAGGPAV, contacts the deazaflavin hydroxyl (obscured), the side chain phospholactate, and the carboxylate of the resolved side chain glutamate and also forms the putative polyglutamate binding cleft (green). (B) A patch of positively charged residues (blue) lines the poly-Glu binding cleft and is surrounded by a more distant ring of negatively charged residues (red). F₄₂₀ is indicated as a stick model (green = carbon, red = oxygen, blue = nitrogen, and orange = phosphorus). Molecular models were visualized with MacPyMOL (http://pymol.org/).

FIG. 5.

SIMBAL analysis of four M. tuberculosis LLM family proteins not found by the positive branch models versus a partition of the LLM (PF00296) family based on the TIGRFAMs-modeled clades of F₄₂₀-producing organisms (Table 2) (positive branch) and all members from non-F₄₂₀-producing organisms (negative branch). Window-length-normalized SIMBAL data are plotted as the maximum scores observed over a range of subsequence window lengths, in essence tracing the highest contour across a triangle plot like the one shown in Fig. 3 (top right).

FIG. 6.

SIMBAL analysis of the M. tuberculosis PPOX family gene Rv2991, using an F₄₂₀ biosynthesis-based partition, indicates two strongly correlated regions. Window-length-normalized SIMBAL data are plotted as the maximum scores observed over a range of subsequence window lengths.

FIG. 7.

(A) Crystal structure of the (dimeric) M. tuberculosis PPOX family protein Rv1155, with (monomeric) FMN bound (blue), showing the locations of SIMBAL peaks 1 and 2 (yellow and orange) (see Fig. 6 and Table 3). FMN binds only weakly to Rv1155, which is a likely F₄₂₀-binding enzyme. Extending downwards from the short FMN side chain is an extended cleft which appears complementary to the much longer F₄₂₀ polyglutamate side chain. (B) The FMN-dependent E. coli PPOX family enzyme PdxH is shown with the homologous regions colored. PdxH binds FMN roughly 1,000 times tighter than Rv1155 and contains a pocket into which the FMN side chain fits snugly, while no extended cleft is apparent. Molecular models were visualized with MacPyMOL (http://pymol.org/).