Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors

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.

Figure 1. Genome structure and sequence analysis of mycobacterial SDRs.

A number of SDRs containing an insertion at the NAD binding site appear in mycobacterial organisms. These SDRs always appear in conjunction with a number of genes that appear to be involved in the biosynthesis of the proposed redox partner mycofactocin. Multiple sequence alignment of the ten crystal structures determined here in comparison with 10 other oxidoreductases of known structure which lack the insertion in the primary sequence. The PDB code for each structure is listed. The structures described here all contain NAD (sequences 1–9) except for 3OEC (sequence 10), whereas for the structures described elsewhere 5 representative examples of NADP (sequences 11–15) or NAD (sequences 16–20) bound structures were selected and the ligand identity is also listed. The magenta cylinders indicate α helices, whereas the blue arrows indicate β strands. The insertion loop is shown as a gray arrow and labeled, and the loop region that is often disordered is also shown as a gray arrow and labeled.

Figure 2. Structural analysis of mycobacterial SDR NAD binding sites.

(A) Carveol dehydrogenase from M. paratuberculosis (MypaA.01326.b) bound to NAD (PDB ID 3PGX). The insertion loop which cover the NAD is shown in green and the loop often disordered in other SDR crystal structure is show in blue (left) and surface rendering showing the available solvent channel with hydrophobic regions shown in white, electropositive regions shown in blue and electronegative regions shown in red (right). (B) SDR from M. abscessus (MyabA.01326.f) bound to NAD (PDB ID 3S55). (C) FabG oxidoreductase from Staphylococcus aureus bound to NADPH (PDB ID 3SJ7). (D) FabG oxidoreductase from Bacillus sp. SG-1 bound to NAD (PDB ID 4NBU). This figure is intended to be representative examples of crystal structures of SDRs with the insertion loop determined here (panels A, B) with those bound to a more open NAD/NADP (panel C) and those bound to a more closed NAD/NADP (panel D); see Table 2 for calculated solvent accessibility of these and other structures.

Figure 3. NMR spectroscopic analysis of mycobacterial SDR ligand binding.

Reference (blue) and saturation (red) STD NMR spectra overlaid for a M. avium SDR (SSGCID ID MyavA.01326.l) in the presence of (A) cofactor NADH (2.1% max STD signal), (B) potential product (S)-(+)-carvone (20.5% max STD NMR signal), (C) potential inhibitor tricyclazol (19.0% max STD NMR signal), and (D) redox partner DCIP (10.2% max STD NMR signal). Although NADH appears to be a non-binder under these conditions, the other compounds all show clear effects of binding.