A phosphoenzyme mimic, overlapping catalytic sites and reaction coordinate motion for human NAMPT.
Nicotinamide phosphoribosyltransferase (NAMPT) is highly evolved to capture nicotinamide (NAM) and replenish the nicotinamide adenine dinucleotide (NAD(+)) pool during ADP-ribosylation and transferase reactions. ATP-phosphorylation of an active-site histidine causes catalytic activation, increasing NAM affinity by 160,000. Crystal structures of NAMPT with catalytic site ligands identify the phosphorylation site, establish its role in catalysis, demonstrate unique overlapping ATP and phosphoribosyltransferase sites, and establish reaction coordinate motion. NAMPT structures with beryllium fluoride indicate a covalent H247-BeF(3)(-) as the phosphohistidine mimic. Activation of NAMPT by H247-phosphorylation causes stabilization of the enzyme-phosphoribosylpyrophosphate complex, permitting efficient capture of NAM. Reactant and product structures establish reaction coordinate motion for NAMPT to be migration of the ribosyl anomeric carbon from the pyrophosphate leaving group to the nicotinamide-N1 while the 5-phosphoryl group, the pyrophosphate moiety, and the nicotinamide ring remain fixed in the catalytic site.
Burgos
Emmanuel S
ES
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Ho
Meng-Chiao
MC
Almo
Steven C
SC
Schramm
Vern L
VL
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D011756:Diphosphates / Q000737:chemistry; D004789:Enzyme Activation; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009038:Motion; D009536:Niacinamide / Q000737:chemistry; D054409:Nicotinamide Phosphoribosyltransferase / Q000737:chemistry*; D010766:Phosphorylation; D013379:Substrate Specificity
D011756:Diphosphates; D009536:Niacinamide; D006639:Histidine; D000255:Adenosine Triphosphate; D054409:Nicotinamide Phosphoribosyltransferase; D000251:Adenosine Triphosphatases