An efficient, multiply promiscuous hydrolase in the alkaline phosphatase superfamily

Abstract

We report a catalytically promiscuous enzyme able to efficiently promote the hydrolysis of six different substrate classes. Originally assigned as a phosphonate monoester hydrolase (PMH) this enzyme exhibits substantial second-order rate accelerations ((k(cat)/K(M))/k(w)), ranging from 10(7) to as high as 10(19), for the hydrolyses of phosphate mono-, di-, and triesters, phosphonate monoesters, sulfate monoesters, and sulfonate monoesters. This substrate collection encompasses a range of substrate charges between 0 and -2, transition states of a different nature, and involves attack at two different reaction centers (P and S). Intrinsic reactivities (half-lives) range from 200 days to 10(5) years under near neutrality. The substantial rate accelerations for a set of relatively difficult reactions suggest that efficient catalysis is not necessarily limited to efficient stabilization of just one transition state. The crystal structure of PMH identifies it as a member of the alkaline phosphatase superfamily. PMH encompasses four of the native activities previously observed in this superfamily and extends its repertoire by two further activities, one of which, sulfonate monoesterase, has not been observed previously for a natural enzyme. PMH is thus one of the most promiscuous hydrolases described to date. The functional links between superfamily activities can be presumed to have played a role in functional evolution by gene duplication.

Fig. 1.

Structures of substrates that are hydrolyzed by BcPMH: 1a: phenyl phosphate, 1b: p-nitrophenyl phosphate, 2a: diphenyl phosphate, 2b: p-nitrophenyl ethyl phosphate, 3b: paraoxon, 4a: phenyl phenylphosphonate, 4b: p-nitrophenyl phenylphosphonate, 5a: phenyl sulfate, 5b: p-nitrophenyl sulfate, 6a: phenyl phenylsulfonate, 6b: p-nitrophenyl phenylsulfonate. Gray lines indicate the bonds that are broken during hydrolysis.

Fig. 2.

pH-rate profiles of k_cat/K_M for all six substrates: (A) phosphate monoester 1b, (B) phosphate diester 2b, (C) phosphate triester 3b, (D) phosphonate monoester 4b, (E) sulfate monoester 5b, and (F) sulfonate monoester 6b. For details see SI Text.

Fig. 3.

X-ray structure of BcPMH. (A) BcPMH is tetrameric in the crystal and in solution. The surface of the active site residues at the bottom of the active site cavity is orange (Upper Left protomer). The arrow indicates the position of the cut for C. (B) Active site residues as identified by homology to RlPMH (11) and P. aeruginosa arylsulfatase. Hydrogen bonds and metal-binding contacts are indicated by dotted lines. The metal ions identified in the protein were iron and zinc. (C) A cut through the active site illustrates the spacious active site opening. (D) Proposed mechanism for all five different conversions involving the fGly nucleophile [on the basis of the proposed mechanism of RlPMH (11)]. Upon nucleophilic attack of fGly on the central P/S atom (X) the leaving group (ROH = phenolate) is abstracted. For all substrates except 3b, a covalent intermediate formed via fGly is broken down by cleaving the same C-O bond (red, instead of a second attack at X).