Divergence of chemical function in the alkaline phosphatase superfamily: structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase - PubMed (original) (raw)

. 2011 May 3;50(17):3481-94.

doi: 10.1021/bi200165h. Epub 2011 Apr 8.

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Divergence of chemical function in the alkaline phosphatase superfamily: structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase

Alexander Kim et al. Biochemistry. 2011.

Abstract

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and (32)P-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

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Figures

Figure 1

Figure 1

(A). The four subunits observed in the asymmetric unit of the crystal of the PAc hydrolase(phosphonoformate complex). The zinc ions are shown as black spheres and the phosphonoformate ligand in red stick. (B). The PAc biological dimer with residues that comprise the subunit-subunit interface shown in black or brown colored stick.

Figure 2

Figure 2

The backbone fold (left panel) and zinc ion-binding site (right panel) of (A.) PAc hydrolase bound with phosphonoformate, (B) NPP bound with AMP and (C) AP bound with orthophosphate. The catalytic domain is colored gray, the cap domain wheat and the linkers pale green. The oxygen atoms are colored red, the nitrogen atoms blue, the phosphorus atoms orange and the carbon atoms green or cyan. The zinc ions are shown as black spheres.

Figure 3

Figure 3

The model of PAc bound to the PAc hydolase active site. The oxygen atoms are colored red, the nitrogen atoms blue, the phosphorus atoms orange and the carbon atoms green. The zinc ions are shown as black spheres.

Figure 4

Figure 4

The Vm (●) and Vm/Km (○) pH rate profiles measured for PAc hydrolase catalyzed hydrolysis of PAc at 25 °C. The Vm/Km data were fitted to equation 3 to define an apparent pK1 and pK2 of 7 ± 2. The Vm pH rate data were fitted to equation 4 to define an apparent pKa = 6.4 ± 0.1.

Figure 5

Figure 5

SDS-PAGE analysis of PAc hydrolase autophosphorylation with [γ 32P]ATP. The top panel is the Coomassie blue stained gel and the bottom panel is the audioradiograph of the gel. Lane 1: Protein molecular weight standards. Lane 2: The reaction of [γ 32P]ATP with acid-denatured wild-type PAc hydrolase. Lane 3: The reaction of [γ 32P]ATP with wild-type PAc hydrolase. Lane 4: The reaction of [γ 32P]ATP with wild-type PAc hydrolase in the presence of 2 mM phosphonoformate. Lane 5: The reaction of [γ 32P]ATP with acid-denatured T64A PAc hydrolase. Lane 6: The reaction of [γ 32P]ATP with T64A PAc hydrolase. Lane 7: The reaction of [γ 32P]ATP with T64A PAc hydrolase in the presence of 2 mM phosphonoformate.

Scheme 1

Scheme 1

The pathways associated with AEP biosynthesis and AEP biodegradation. Abbreviations used are PEP (phosphoenol pyruvate), PPyr (phosphonopyruvate), PAld (phosphonoacetaldehyde), Glu (L-glutamate), αKG (α-ketoglutarate), Pyr (pyruvate), Ala (L-alanine) and PAc (phosphonoacetate).

Scheme 2

Scheme 2

The mechanism for phosphonatase catalyzed hydrolysis of PAld (–18).

Scheme 3

Scheme 3

A proposed model for the catalytic mechanism used by PAc hydrolase in the first partial reaction.

Chart 1

Chart 1

The structures of the compounds tested as substrate or inhibitor.

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