Substrate-promoted formation of a catalytically competent binuclear center and regulation of reactivity in a glycerophosphodiesterase from Enterobacter aerogenes

Abstract

The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous binuclear metallohydrolase that catalyzes the hydrolysis of mono-, di-, and triester substrates, including some organophosphate pesticides and products of the degradation of nerve agents. GpdQ has attracted recent attention as a promising enzymatic bioremediator. Here, we have investigated the catalytic mechanism of this versatile enzyme using a range of techniques. An improved crystal structure (1.9 A resolution) illustrates the presence of (i) an extended hydrogen bond network in the active site, and (ii) two possible nucleophiles, i.e., water/hydroxide ligands, coordinated to one or both metal ions. While it is at present not possible to unambiguously distinguish between these two possibilities, a reaction mechanism is proposed whereby the terminally bound H2O/OH(-) acts as the nucleophile, activated via hydrogen bonding by the bridging water molecule. Furthermore, the presence of substrate promotes the formation of a catalytically competent binuclear center by significantly enhancing the binding affinity of one of the metal ions in the active site. Asn80 appears to display coordination flexibility that may modulate enzyme activity. Kinetic data suggest that the rate-limiting step occurs after hydrolysis, i.e., the release of the phosphate moiety and the concomitant dissociation of one of the metal ions and/or associated conformational changes. Thus, it is proposed that GpdQ employs an intricate regulatory mechanism for catalysis, where coordination flexibility in one of the two metal binding sites is essential for optimal activity.

Figure 1

Schematic representation of the active site of GpdQ based on the improved crystallographic data presented here. In two of the six active sites an additional water molecule is also seen bound terminally to the β metal and further hydrogen bonded to the amine nitrogen on N80.

Figure 2

MCD spectra of wild-type GpdQ at 1.45 K, 3.5 T depicting a mononuclear Co^II center, dinuclear Co^II center and a dinuclear Co^II center in the presence of phosphate.

Figure 3

pH dependence of the activity (k_cat) and catalytic efficiency (k_cat/K_M) of the hydrolysis of pNPP and EtpNPP by Co^II-substituted GpdQ. The arrows indicate the pH values used for KIE measurements (see below).

Figure 4

MCD spectra of Co^II-substituted N80A and N80D mutants of GpdQ. Note that for N80A-GpdQ in the absence of phosphate up to 100 equivalents of Co^II were added, but only the transition at 495 nm was observed.

Figure 5

The structure of pNPP and EtpNPP, and the isotope effects determined.

Scheme 1

Scheme for the proposed reaction mechanism for GpdQ-catalyzed hydrolysis.