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 Å 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.

Spectroscopic and mechanistic studies of dinuclear metallohydrolases and their biomimetic complexes

“…The two metal binding sites are often defined as the α-and β-site, differentiated by their coordination environments. 38 In this review we focus on the dinuclear metalloenzymes OpdA and, in particular, GpdQ. OpdA is an extremely efficient phosphotriesterase with low levels of activity towards phosphodiesters.…”

“…41,42 A structure with higher resolution (1.9 Å) is, however, now available. 38,75 The structure shows that GpdQ is a hexamer which consists of a trimer of dimers, having overall D 3 symmetry. 38 Fig .…”

“…38,75 The structure shows that GpdQ is a hexamer which consists of a trimer of dimers, having overall D 3 symmetry. 38 Fig . 4 displays the hexamer (a) and the structure of the dimeric subunit (b).…”

“…The active site in the resting state is mononuclear with only the six-coordinate α-site being occupied by a metal ion. 38 In an attempt to evolve GpdQ towards the non-physiological substrate bis(4-nitrophenyl)phosphate (BPNPP) a range of mutants were obtained by directed evolution. 76 These mutants exhibited increased activity towards BPNPP and the previously found hexameric structure was broken down in some mutants.…”

“…76 The more recent crystal structure at higher resolution illustrates an extensive hydrogen bonding network in the metal binding active site connecting it with the second coordination sphere. 38,75 The α-site features four amino acid side chains, two aspartates (Asp50, Asp8) and two histidines (His10, His197), whereas the metal in the β-site, vacant in the resting state, is coordinated by two histidines (His156, His195), one aspartate (Asp50) and one asparagine (Asn80). 75,77,78 The crystal structure also suggests the presence of a terminal H 2 O Fig.…”