title: Nicotinamide mononucleotide adenylyltransferase displays alternate binding modes for nicotinamide nucleotides.
abstract: Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the biosynthesis of NAD(+) and NaAD(+). The crystal structure of NMNAT from Methanobacterium thermoautotrophicum complexed with NAD(+) and SO4(2-) revealed the active-site residues involved in binding and catalysis. Site-directed mutagenesis was used to further characterize the roles played by several of these residues. Arg11 and Arg136 were implicated in binding the phosphate groups of the ATP substrate. Both of these residues were mutated to lysine individually. Arg47 does not interact with either NMN or ATP substrates directly, but was deemed to play a role in binding as it is proximal to Arg11 and Arg136. Arg47 was mutated to lysine and glutamic acid. Surprisingly, when expressed in Escherichia coli all of these NMNAT mutants trapped a molecule of NADP(+) in their active sites. This NADP(+) was bound in a conformation that was quite different from that displayed by NAD(+) in the native enzyme complex. When NADP(+) was co-crystallized with wild-type NMNAT, the same structural arrangement was observed. These studies revealed a different conformation of NADP(+) in the active site of NMNAT, indicating plasticity of the active site.
authors.affiliation: Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.
authors.forename: Roland
authors.initials: R
authors.lastname: Pfoh
authors.affiliation: Campbell Family Institute for Cancer Research, Princess Margaret Cancer Center, University Health Network, Toronto Medical Discovery Tower-MaRS Centre, 101 College Street, Toronto, ON M5G 1L7, Canada.
authors.forename: Emil F
authors.initials: EF
authors.lastname: Pai
authors.affiliation: Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.
authors.forename: Vivian
authors.initials: V
authors.lastname: Saridakis
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D017014:Methanobacterium / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D009243:NAD; D009249:NADP; D000255:Adenosine Triphosphate; D009612:Nicotinamide-Nucleotide Adenylyltransferase
keywords: Methanobacterium thermoautotrophicum; NAD biosynthesis; NMN adenylyltransferase; protein crystallography; structural biology