Abstract
During human infection, Mycobacterium tuberculosis (Mtb) survives the normally bacteriocidal phagosome of macrophages. Mtb and related species may be able to combat this harsh acidic environment which contains reactive oxygen species due to the mycobacterial genomes encoding a large number of dehydrogenases. Typically, dehydrogenase cofactor binding sites are open to solvent, which allows NAD/NADH exchange to support multiple turnover. Interestingly, mycobacterial short chain dehydrogenases/reductases (SDRs) within family TIGR03971 contain an insertion at the NAD binding site. Here we present crystal structures of 9 mycobacterial SDRs in which the insertion buries the NAD cofactor except for a small portion of the nicotinamide ring. Line broadening and STD-NMR experiments did not show NAD or NADH exchange on the NMR timescale. STD-NMR demonstrated binding of the potential substrate carveol, the potential product carvone, the inhibitor tricyclazol, and an external redox partner 2,6-dichloroindophenol (DCIP). Therefore, these SDRs appear to contain a non-exchangeable NAD cofactor and may rely on an external redox partner, rather than cofactor exchange, for multiple turnover. Incidentally, these genes always appear in conjunction with the mftA gene, which encodes the short peptide MftA, and with other genes proposed to convert MftA into the external redox partner mycofactocin.
Highlights
Redox enzymes that bind a single NAD cofactor can act in several conceptually different ways
Based off genomic analysis we recently identified a widely distributed set of three uncharacterized genes, found in Mycobacterium tuberculosis and dozens of other species, and proposed that this system is responsible for the biosynthesis of a peptide-derived natural product we named mycofactocin which is similar in function to PQQ5
69 mycobacterial dehydrogenases from TIGR03971 with an insertion near the proposed cofactor binding site were entered into the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure determination pipeline[9]
Summary
Redox enzymes that bind a single NAD cofactor can act in several conceptually different ways. In scheme A, the enzyme can oxidize a substrate by reducing NAD+to NADH, and allow both the product and the cofactor to diffuse away. In scheme B, the NAD cofactor remains tightly bound, and no net redox change to the substrate occurs. In scheme C, the NAD cofactor remains tightly bound and does not dissociate from the enzyme after the physiological substrate undergoes a net redox change; instead electron transfer occurs between a bound NAD+and free NADH. It achieves multiple turnovers, despite its non-exchangeable cofactor, by tying net oxidation of one substrate to net reduction of another. The three genes proposed to be involved in mycofactocin biosynthesis always appear together or not at all: mftA encodes a short peptide with a C-terminal sequence IDGXCGVY, mftB encodes a peptide chaperone, and mftC encodes a radical S-adenosyl methionine enzyme (rSAM). MftC was shown to catalyze the decarboxylation of the C-terminal tyrosine of MftA in the presence of MftB, presumably the first step in the biosynthetic pathway of mycofactocin
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