Redesign of Schistosoma mansoni NAD+ catabolizing enzyme: active site H103W mutation restores ADP-ribosyl cyclase activity.
Schistosoma mansoni NAD(P)+ catabolizing enzyme (SmNACE) is a new member of the ADP-ribosyl cyclase family. In contrast to all the other enzymes that are involved in the production of metabolites that elicit Ca2+ mobilization, SmNACE is virtually unable to transform NAD+ into the second messenger cyclic ADP-ribose (cADPR). Sequence alignments revealed that one of four conserved residues within the active site of these enzymes was replaced in SmNACE by a histidine (His103) instead of the highly conserved tryptophan. To find out whether the inability of SmNACE to catalyze the canonical ADP-ribosyl cyclase reaction is linked to this change, we have replaced His103 with a tryptophan. The H103W mutation in SmNACE was indeed found to restore ADP-ribosyl cyclase activity as cADPR amounts for 7% of the reaction products (i.e., a value larger than observed for other members of this family such as CD38). Introduction of a Trp103 residue provides some of the binding characteristics of mammalian ADP-ribosyl cyclases such as increased affinity for Cibacron blue and slow-binding inhibition by araF-NAD+. Homology modeling of wild-type and H103W mutant three-dimensional structures, and docking of substrates within the active sites, provides new insight into the catalytic mechanism of SmNACE. Both residue side chains share similar roles in the nicotinamide-ribose bond cleavage step leading to an E.ADP-ribosyl reaction intermediate. They diverge, however, in the evolution of this intermediate; His103 provides a more polar environment favoring the accessibility to water and hydrolysis leading to ADP-ribose at the expense of the intramolecular cyclization pathway resulting in cADPR.
Kuhn
Isabelle
I
Institut Gilbert Laustriat, UMR 7175 CNRS, Université Louis Pasteur (Strasbourg I), Faculté de Pharmacie, 67401 Illkirch, France.
Kellenberger
Esther
E
Rognan
Didier
D
Lund
Frances E
FE
Muller-Steffner
Hélène
H
Schuber
Francis
F
D036541:ADP-ribosyl Cyclase / Q000235:genetics* / Q000378:metabolism; D051997:ADP-ribosyl Cyclase 1 / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D036563:Cyclic ADP-Ribose / Q000235:genetics / Q000378:metabolism; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D009243:NAD / Q000378:metabolism*; D012550:Schistosoma mansoni / Q000201:enzymology / Q000235:genetics*
D015801:Helminth Proteins; D009243:NAD; D036563:Cyclic ADP-Ribose; D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D002118:Calcium