Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

Abstract

SummaryThe e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD + utilization pathway by dephosphorylating NMN to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases, which are nonspecific 5′-, 3′-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with NMN, 5′-AMP, 3′-AMP, and 2′-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and hydrogen-bonding edge of the base. The span between the hydrophobic box and phosphoryl site is optimal for recognizing nucleoside monophosphates, which explains the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, which is consistent with observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5′-and 3′-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5′ substrates in an anti conformation and 3′ substrates in a syn conformation. Finally, the structures suggest that class B and C acid phosphatases share a common strategy for nucleotide recognition.

Crystal structure and immunogenicity of the class C acid phosphatase from Pasteurella multocida

“…Structures of rP4 have shown that the lower part of the active site contains the substrate phosphoryl binding site, which includes an essential Mg 2+ ion surrounded by the four Asp residues of the DDDD motif [9, 15]. The roles of these Asp residues in rPmCCAP can be inferred from structures of rP4 complexed with substrates [15] and more generally, studies of HAD superfamily enzymes [27]. The first Asp (Asp63 in rPmCCAP, Fig.…”

“…The estimated free energy of dissociation from PISA (42 kcal/mol) suggests that the dimer is stable in solution. Furthermore, an identical dimer is found in both the tetragonal and hexagonal rP4 crystal lattices [9, 15], and several intersubunit interactions are observed in both enzymes. These conserved interactions include an intersubunit Glu-Arg-Asn electrostatic network that involves an active site loop (Fig.…”

“…4B). Previous structures of a substrate-trapping mutant of rP4 complexed with nucleoside monophosphates showed that the base of the substrate binds in an aromatic box formed by Phe86, Trp91, and Tyr221 [15]. The analogous residues of rPmCCAP are Phe85, Trp90, and Tyr220, and they form a substructure identical to that of rP4.…”

“…Because of their localization to the bacterial outer membrane, CCAPs are potential candidates for vaccine development, and some progress has been made toward creating a vaccine against nontypeable Haemophilus influenzae using catalytically inactive mutants of the H. influenzae CCAP [2–4]. Biochemical studies have shown that CCAPs typically hydrolyze a variety of aryl phosphatases as well as nucleoside 5´- and 3´-monophosphates [5–15]. Crystallographic studies of the recombinant CCAP from H. influenzae , a.k.a.…”

“…Crystallographic studies of the recombinant CCAP from H. influenzae , a.k.a. rP4, have shown that CCAPs belong to the haloacid dehalogenase (HAD) structural superfamily and revealed the basis of selectivity for nucleoside monophosphate substrates [9, 15]. The structural and biochemical data also showed that the first of the four conserved Asp residues is the nucleophile that attacks the P atom of the substrate, the second one functions as the acid that protonates the leaving group, and all four Asp residues contribute to the binding of a Mg 2+ ion.…”