Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate

Abstract

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 10(5), suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.

Figure 1

Consensus motif for the PLD superfamily. (A) The conserved H, K, and D residues are boxed. (B) SDS gel electrophoresis of proteins present in the bacterial extracts (lane 1), proteins present after induction of wild-type Nuc, the K96S and H94N mutants of Nuc (lanes 2–4, respectively), and the corresponding purified proteins (lanes 5–7).

Figure 2

DEPC inactivation of Nuc. (A) Inactivation of Nuc by increasing concentrations of DEPC. Nuc (12 μM) was incubated for 5 min at pH 4.5 with 50 mM DEPC. Hydroxylamine was added to one-half of the reaction at a final concentration of 0.5 M. At the indicated times, the reaction mixtures were diluted 10-fold into assay buffer containing 100 mM pNP-PPh at pH 7.0. Activity was measured for 1 min as described in Experimental Procedures. (B) Time course of inactivation, in presence and absence of tungstate. Nuc (12 μM) was mixed with 50 mM DEPC in the presence or absence (“Control”) of 1 mM tungstate at pH 4.5. Aliquots were removed at the indicated times and diluted 10-fold into assay buffer containing 100 mM pNP-PPh, at pH 7.0, and activity was measured as described.

Scheme 1

Scheme 2

Figure 3

Formation and analysis of a covalent phosphoenzyme intermediate. (A) Wild-type, K96S, H94N, H140N, or S109A Nuc (50 μM) was mixed with 1 mM [³²P]phosphate at pH 4.5 and incubated for the indicated times at room temperature, where upon the reactions were quenched and subjected to SDS/PAGE and autoradiography. (B) Nuc was phosphorylated for 1 min with [³²P]phosphate, followed by the addition of 100 mM unlabeled potassium phosphate. The reaction was quenched at the indicated time points, and samples were subjected to SDS/PAGE and autoradiography.

Figure 4

(A) Chemical properties of the phosphoenzyme intermediate. ³²P-labeled, phosphorylated Nuc was transferred to Immobilon P. Filter strips were counted, then incubated in: (i) 1 M HCl; (ii) 1 M NaOH; (iii) 1 M hydroxylamine in 200 mM Tris⋅Cl, pH 7.4; and (iv)1 M Tris⋅Cl, pH 7.4, for 45 min at 37°C. Radioactivity remaining on each filter strip was visualized by autoradiography and quantitated by counting. (B) Filter strips were incubated in 50 mM sodium citrate, pH 2.5, or in solutions of 25 mM Tris, 25 mM Bis·Tris, and 50 mM acetic acid at pH values ranging from 3.5 to 8.8, for 30 min at 80°C. The amount of radioactivity on each filter strip before and after incubation was quantitated by scintillation spectrometry.

Figure 5

Phosphoamino acid analysis. Chromatography of ³²P-labeled amino acids after alkaline hydrolysis of ³²P-labeled endonuclease. The position of inorganic phosphate (Pi), phosphoarginine, phospholysine, phosphothreonine, phosphohistidine, and phosphotyrosine are noted on the chromatogram. Phosphoserine elutes 30 sec after phosphothreonine (11). Fluorescence is plotted on the right axis and is represented by a continuous line. Radioactivity is plotted on the left axis and is represented by the data points.