Redesign of Schistosoma mansoni NAD+ Catabolizing Enzyme: Active Site H103W Mutation Restores ADP-Ribosyl Cyclase Activity

Abstract

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.