The crystal structure of Bacillus cereus phosphonoacetaldehyde hydrolase: Insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofact or. The reaction proceeds via a novel bicovalent catalytic mechanism in whi ch an active-site nucleophile abstracts the phosphoryl group from the Schif f-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this st udy, the X-ray crystal structure of the Bacillus cereus phosphonatase homod imer complexed with the phosphate (product) analogue tungstate (K-i = 50 mu M) and the Mg(II) cofactor was determined to 3.0 Angstrom resolution with an R-cryst = 0.248 and R-free = 0.284. Each monomer is made up of an alpha/ beta core domain consisting of a centrally located six-stranded parallel be ta-sheet surrounded by six alpha-helices. Two flexible, solvated linkers co nnect to a small cap domain (residues 21-99) that consists of an antiparall el, five-helix bundle. The subunit-subunit interface, formed by the symmetr ical packing of the two alpha 8 helices from the respective core domains, i s stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp 164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interface of each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg( II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186. the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond w ith the carboxylate of Asp190 and Thr187. The guanidinium group of Arg160 b inds tungstate and the proposed nucleophile Asp12, which is suitably positi oned for in-line attack at the tungsten atom. The side chains of the core d omain residue Tyr128 and the cap domain residues Cys22 and Lys53 are locate d nearby. The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp1 2Ala substitution. The similarity of backbone folds observed in phosphonata se and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conserved a ctive-site scaffold having distinct catalytic stations. Analysis of the usa ge of polar amino acid residues at these stations by the dehalogenases, pho sphonatases, phosphatases, and phosphomutases of the HAD superfamily sugges ts possible ways in which the active site of an ancient enzyme ancestor mig ht have been diversified for catalysis of C-X, P-C, and P-O bond cleavage r eactions.