Abstract
The crystal structure of the alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei was solved in the presence of three inhibitors: acarbose, Tris, and zinc. In the absence of exogenous metals, this alpha-amylase bound 1 and 4 molar eq of zinc and calcium, respectively. The structure reveals a novel, activating, two-metal (Ca,Zn)-binding site and a second inhibitory zinc-binding site that is found in the -1 sugar-binding pocket within the active site. The data resolve the apparent paradox between the zinc requirement for catalytic activity and its strong inhibitory effect when added in molar excess. They provide a rationale as to why this alpha-amylase, in contrast to commercially available alpha-amylases, does not require the addition of metal ions for full catalytic activity, suggesting it as an ideal target to maximize the efficiency of industrial processes like liquefaction of starch.
Highlights
␣-Amylases (␣-1,4-D-glucan 4-glucanohydrolase, EC 3.2.1.1) are endo-acting hydrolases that randomly cleave the ␣-1,4glycosidic linkages of branched and linear carbohydrates such as amylopectin and amylose in starch and glycogen [1,2,3]
The structures are in the presence of three different inhibitors: 1) zinc (PWA1⁄7Zn); 2) the specific substrate analog acarbose and zinc (PWA1⁄7Ac/Zn); and 3) Tris, which was used as a buffer for crystallization trials in the absence of zinc (PWA1⁄7Tris)
We initially identified the nature of the bound metal ions by proton-induced x-ray emission (PIXE)
Summary
Gene Amplification and Expression—The wild-type gene encoding PWA was amplified by PCR using primers deduced from the open reading frame of the P. furiosus wild-type ␣-amylase gene [10]. A heavy atom derivative suitable for crystallographic phase determination was obtained by soaking PWA1⁄7Zn in a solution containing 29% (v/v) polyethylene glycol monomethyl ether 550 and 1.0 mM CH3HgCl for 1.5 h at 19 °C prior to x-ray data collection. X-ray data sets for the PWA1⁄7Zn, PWA1⁄7Ac/Zn, and PWA1⁄7Tris complexes were recorded up to 2.2-, 2.0-, and 1.5-Å resolution, respectively. Metals were refined as zinc when the crystal growth conditions contained zinc ions and if the metals sites showed a high anomalous peak emerging from data set collection at 12.4 keV ( ϭ 0.91 Å), which is above the k-adsorption edge of zinc of 9.59 keV ( ϭ 1.28 Å)
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