title: 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.
authors.affiliation: National Institutes of Health (NIH), Bethesda, MD 20892, USA.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Haft
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Beryllium Discovery Corp., 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
authors.forename: Phillip G
authors.initials: PG
authors.lastname: Pierce
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Beryllium Discovery Corp., 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Mayclin
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Beryllium Discovery Corp., 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Sullivan
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Beryllium Discovery Corp., 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
authors.forename: Anna S
authors.initials: AS
authors.lastname: Gardberg
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Beryllium Discovery Corp., 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
authors.forename: Jan
authors.initials: J
authors.lastname: Abendroth
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Beryllium Discovery Corp., 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
authors.forename: Darren W
authors.initials: DW
authors.lastname: Begley
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), 307 Westlake Avenue North, Seattle WA 98109, USA.
authors.forename: Isabelle Q
authors.initials: IQ
authors.lastname: Phan
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), 307 Westlake Avenue North, Seattle WA 98109, USA.
authors.forename: Bart L
authors.initials: BL
authors.lastname: Staker
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), 307 Westlake Avenue North, Seattle WA 98109, USA.|University of Washington, Department of Medical Education and Biomedical Informatics &Department of Global Health, Seattle WA 98195, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Myler
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Beryllium Discovery Corp., 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
authors.forename: Vasilios M
authors.initials: VM
authors.lastname: Marathias
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Beryllium Discovery Corp., 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
authors.forename: Donald D
authors.initials: DD
authors.lastname: Lorimer
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA 98109, USA.|Beryllium Discovery Corp., 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Edwards
mesh_terms: D015086:2,6-Dichloroindophenol / Q000378:metabolism; D001665:Binding Sites; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D000081005:Cyclohexane Monoterpenes; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D039821:Monoterpenes / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009243:NAD / Q000737:chemistry* / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013844:Thiazoles / Q000378:metabolism
chemical_list: D003067:Coenzymes; D000081005:Cyclohexane Monoterpenes; D039821:Monoterpenes; D013844:Thiazoles; D009243:NAD; C006923:carvone; C075856:carveol; D015086:2,6-Dichloroindophenol; D010088:Oxidoreductases; C041191:tricyclazole