Investigation of Metal Ion Binding in Phosphonoacetaldehyde Hydrolase Identifies Sequence Markers for Metal-Activated Enzymes of the HAD Enzyme Superfamily^,

Abstract

The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies. HAD members conserve an R, -core domain that frames the four-loop active-site platform. Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer). In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members. Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor. Analysis of the minimum requirements for metal cofactor binding was carried out using Mg-(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members. Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate. The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand. X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate. Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it. Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue. Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.In this paper, we examine metal ion activation of phosphonoacetaldehyde hydrolase (phosphonatase 1 )-catalyzed hydrolysis of phosphonoacetaldehyde (Pald) to orthophosphate and acetaldehyde (Figure 1). Phosphonatase is a member of the 2-haloalkanoate dehalogenase (HAD) enzyme superfamily (1, 2). This is an exceptionally large family of enzymes (∼2700 members), which includes carbon (2-haloalkonoate dehalogenases) and phosphorus hydrolases (ATPases, phosphate monoesterases, and hexose phosphate mutases) (3). The members of this family catalyze nucleophilic substitution reactions at phosphorus or carbon centers, using a conserved Asp carboxylate in covalent catalysis. Like phosphonatase (4), most, but not all, members of the superfamily possess a cap domain, the fold and function of which are variable. The

The Structure of a Cyanobacterial Sucrose-Phosphatase Reveals the Sugar Tongs That Release Free Sucrose in the Cell

“…Several of the conserved residues in motifs I and III of many HAD superfamily proteins are involved in the coordination shell of a divalent metal ion cofactor (Zhang et al, 2004). In the active site of the open conformation of the SPP structure, we observed a high electron density that we interpret to be a Mg 2þ ion, given the enzyme's dependence on Mg 2þ for catalytic activity and its high affinity for this metal ion (the activation constant is 70 mM; Lunn, 2002).…”

“…Presumably, the negatively charged phosphate group of Suc6P would attract the Mg 2þ ion, and substrate binding might also indirectly favor movement of the Mg 2þ ion into the catalytic position by inducing conformational changes in the active site. The altered position of the Mg 2þ ion in the D12A mutant of the Bacillus cereus phosphonatase suggests that the nucleophilic Asp residue has a strong influence on the position of the metal ion (Zhang et al, 2004). In the crystals of SPP soaked with Suc6P ( Figure 4C), we observed only sucrose and phosphate bound at the active site, indicating that the Suc6P had been completely dephosphorylated.…”

“…The reaction mechanisms of several HAD superfamily enzymes have been studied in detail. All of them catalyze a nucleophilic substitution reaction at phosphorus or carbon centers using a conserved Asp carboxylate in covalent catalysis, and most contain a metal cofactor (Zhang et al, 2004). Based on this information, and comparison of the SPP crystal structures with other known HAD protein structures, we propose the following two-step catalytic mechanism for SPP.…”

Interaction of Genome-linked Protein (VPg) of Turnip Mosaic Virus with Wheat Germ Translation Initiation Factors eIFiso4E and eIFiso4F

“…This suggests that these proteins use different catalytic mechanisms to catalyze the same reaction. The molecular mechanisms of this catalytic reaction have been characterized only for HAD-like phosphohydrolases (36,37), which cleave covalent bonds of phosphorylated substrates by nucleophilic attack of the motif I Asp on the phosphorus of the substrate, resulting in the formation of a phosphoenzyme intermediate. In HAD-like phosphohydrolases, the metal cofactor (usually Mg 2ϩ ) is involved in the coordination of the nucleophilic side-chain carboxylate of catalytic Asp and the phosphoryl group of the substrate (36,37).…”

The catalytic scaffold of the haloalkanoic acid dehalogenase enzyme superfamily acts as a mold for the trigonal bipyramidal transition state

“…Earlier work showed that substrate-recognition elements and chemical-catalysis elements are separately located (4)(5)(6)(7)(8)(9). Specifically, substrate recognition is delegated to residues located outside of the core domain active site cleft, whereas phosphoryl transfer is mediated by residues located deep inside this cleft.…”