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.

FIGURE 1

(A) Minimal kinetic mechanism for reactions catalyzed by ADP-ribosyl cyclase family members. (B) Molecular mechanism of the reaction catalyzed by SmNACE. This hypothetical mechanism was adapted from studies on mammalian CD38. The reaction involves the cleavage of the nicotinamide-ribosyl bond via a late transition state leading to the formation of either a covalent acylal ADP-ribosyl intermediate or a carboxylate-stabilized oxocarbenium ion intermediate. In either case the intermediate is then transformed via competing pathways : i) reaction with water yielding ADP-ribose, or ii) intramolecular cyclization to form cyclic ADP-ribose by reaction of N1 of the adenine ring with the C1’’ of the intermediate. The catalytic carboxylate corresponds to Glu²⁰² in SmNACE. At a molecular/structural level, the precise mechanism by which the ‘signature’ Glu¹²⁴, the second active site carboxylic group, controls the cyclization to hydrolysis ratio remains poorly understood. For a full discussion of the molecular mechanism of these enzymes, see (3).

FIGURE 2

Sequence alignment between S. mansoni NACE and representative members of the ADP-ribosyl cyclase family. The amino acid sequence alignment (Clustal W 1.82) of the active site domains reveals the conservation of the invariant catalytic Glu (Glu²⁰² ; bold) and other important residues of the active site such as Glu¹²⁴ (bold) and Trp¹⁶⁵ (shaded). The second invariant Trp residue present in the active site of all other members of the cyclase family (grey shaded) is replaced by His¹⁰³ in SmNACE. The ‘signature region’ TLEDTLLGY (shaded) is present in all known CD38 orthologs.

FIGURE 3

Recombinant soluble H103W SmNACE catalyzed transformation of NAD⁺. (A) Analysis by SDS-PAGE and Western blotting of purified recombinant H103W SmNACE mutant. Left lane: silver-stained SDS-PAGE gel (12%) – Right lane: immunoblot using anti-NACE antibody (16). The molecular weights (kDa) of the purified mutant are indicated. (B) Kinetics of NAD⁺ transformation catalyzed by H103W SmNACE mutant. Assays were carried out at 37°C in 10 mM potassium phosphate buffer, pH 7.4. Initial rates were determined at the given substrate concentrations and the data were fitted to the Michaelis-Menten equation. (C) Representative HPLC radiochromatograms of the reaction products observed after transformation of ¹⁴C-labeled NAD⁺ by WT (upper panel) and H103W mutant SmNACE (lower panel). The assays were performed at 37°C in 10 mM potassium phosphate buffer, pH 7.4, in the presence of [¹⁴C]NAD⁺ and the reaction products were monitored by radiodetection. (D) Representative HPLC elution profile of the products obtained by solvolysis of NAD⁺ catalyzed by the H103W SmNACE mutant in the presence of 3.0 M methanol. The methanolysis peak was identified by co-elution with an authentic sample of β-methyl ADPR (57).

FIGURE 4

Effect of methanol, Cibacron blue F3GA and pH on reactions catalyzed by SmNACE. (A) Effect of increasing concentrations of methanol on the rate of transformation of 100 μM NGD⁺ into cGDPR catalyzed by WT (squares) and H103W mutant (triangles) of SmNACE. The assays were carried out at 37°C in 10 mM potassium phosphate buffer, pH 7.4, and the cyclization reaction progress monitored fluorometrically at λ_em = 410 nm (λ_exc = 310nm). Residual activities (means ± SD, n=4) are percent of the activity measured in the absence of solvent. (B) Effect of methanol on the solvolysis/cyclization ratio ( = [GDPR + β-methyl GDPR]/[cGDPR]) observed on transformation of 100 μM NGD⁺ catalyzed by WT SmNACE (means ± SD, n=3). The reaction products were analyzed by HPLC and their relative proportions were quantified as described previously (8). (C) Inhibition of WT and H103W SmNACE by Cibacron blue F3GA. The effect of the Cibacron blue on the initial rates of the transformation of 20 μM 1,N⁶-etheno-NAD⁺ catalyzed by SmNACE in 10 mM potassium phosphate buffer, pH 7.4, was followed fluorometrically (λ_em = 410 nm; λ_exc = 310 nm) at 37°C. The IC₅₀ values were calculated from the plot of residual activity against log of dye concentration. WT (triangles) and H103W SmNACE mutant (squares). (D) pH dependence of WT SmNACE. The pH profile of V_max, measured at 37°C, using 1,N6-etheno-NAD⁺ as substrate displayed a critical ionization with a pKa of 5.82 ± 0.07 which must be unprotonated for catalytic activity. (E) pH profiles of V_max/K_m values determined at 37°C using 1,N⁶-etheno-NAD⁺ as substrate (n = 3). WT (triangles) and H103W SmNACE mutant (squares). In (D) and (E) the solid lines represent the fit of the data (n = 3) to a single pKa model.

FIGURE 5

Three-dimensional structure of WT and H103W SmNACE active sites. Free WT (A) and mutant (B) enzymes. Yellow ribbons illustrate the secondary structure elements that form the catalytic cleft. Side chains of the key residues are depicted using ball-and-stick representation and colored by atom type. NAD⁺ docked into the active site of WT (C) and mutant (D) enzymes. The best GOLD poses (fitness values of 64 and 82, respectively) of NAD⁺ are shown in ball-and-stick and colored by atom type. The active site is represented as Connolly surface and colored according to the electrostatic potential computed using AMBER7 FF99 charges. The color ramp of electrostatic potential (in kcal/mol) ranges from red (most positive) to purple (most negative). The protein is always displayed in the same orientation. The rendering was performed using SYBYL ver. 7.1. Color code : white = C, red = O, dark blue = N, cyan = H and orange = P.

FIGURE 6

Views of cADPR and cGDPR three-dimensional structure. (A) Free cADPR and cGDPR. Best-fit superposition of the pyrophosphate and ribose heavy atoms of the MacroModel lowest energy conformers. For sake of clarity, ribose and pyrophosphate groups are depicted without hydrogen atoms using line representation whereas all atoms of adenine and guanine bases are shown using ball-and-stick representation. Molecules are colored by atom type as defined in Figure 5. H-bond donors and acceptors in the Watson Crick edge of the bases are indicated using arrows. cADPR (B) and cGDPR (C) docked into the WT and H103W SmNACE active site. Carbon atoms of the WT and mutant complexes are shown in grey and green respectively (the other atoms are colored as defined in Figure 4). All ligand heavy atoms are shown using ball-and-stick. Side chains of the key residues involved in ligand binding are depicted using a line representation and illustrate the surrounding enzyme structure. The rendering was performed using SYBYL 7.1.

FIGURE 7

Interaction of the NMN⁺ moiety of NAD⁺ docked into the active site of WT (A) and H103W mutant (B) of SmNACE. Heavy atoms of NMN⁺ and side chains of the enzyme key residues are depicted using ball-and-stick representation. Carbon atoms of NMN⁺ are colored in green, all the other atoms are colored by atom type as defined in Figure 5. The rendering was performed using SYBYL 7.1. The ‘catalytic’ Glu²⁰² is believed to be involved in the stabilization of the putative oxocarbenium ion transition state formed during the nicotinamide-ribosyl bond cleavage and in the stabilization of the ADP-ribosyl reaction intermediate, see (3). The ‘signature’ Glu¹²⁴ is taking part in the partitioning of the intermediate between cyclization and hydrolysis.