Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

Abstract

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the a-amylase (AHA) secreted by this bacterium, in its native state to 2.0 8, resolution as well as in complex with Tris to 1.85 8, resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known a-amylases of various origins with a surprisingly greatest similarity to mammalian a-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate a-1,4-glycosidic bonds. The chloride binding site is situated -5 8, from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found -22 A from the active site. We found that this triad is equally present in other chloride dependent a-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted a-amylase.Keywords: allosteric activation; cold adaptation; crystal structure; glycosyl hydrolases; inhibition; psychrophilic a-Amylases a-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1 are endoglucanases widely distributed in bacteria, fungi, animals, and plants. They catalyze the hydrolysis of starch, glycogen, and related polysaccharides by cleaving internal a-1,Cglycosidic bonds. In addition to their biochemical interest, a-amylases have a number of important biotechnological applications in food and starch processing industries (Vihinen & Mantsala, 1989).Alteromonas haloplanctis is a gram negative bacterium collected in Antarctica and secreting a calcium-and chloride-dependent a-amylase. It grows at sub-zero temperatures in its natural environment and can be considered as an extreme psychrophile (Feller et al., 1992). The psychrophilic a-amylase is characterized by a high catalytic efficiency which compensates for the reduction of chemical reaction rates inherent to low temperatures. In addition, all biophysical parameters recorded suggest a flexible conformation of this heat-labile a-amylase. The two crystal structures reported here are the first three-dimensional structures of a psy- chrophilic enzyme; moreover, they provide a number of significant features related to various aspects of biochemistry and microbiology. The structure of A. haloplanctis a-amylase cornplexed with Tris allows to understand the molecular basis of glycosidase inhibition by this cation which is a widely used compound in biochemistry. This bacterial a-amylase requires a chloride ion for its activity, a property previously considered as specific to mammalian a-amylases (Brayer et al., 1995). ...

Differential Regulation of a Hyperthermophilic α-Amylase with a Novel (Ca,Zn) Two-metal Center by Zinc

“…3, A and B; and 4C (Fig. 3, C and D), confirming previous structural data from a number of ␣-amylase⅐Tris complexes (32, 34 -36) and demonstrating its function as a potent competitive inhibitor (34,37,38).…”

Tyrosine 105 and Threonine 212 at Outermost Substrate Binding Subsites –6 and +4 Control Substrate Specificity, Oligosaccharide Cleavage Patterns, and Multiple Binding Modes of Barley α-Amylase 1

“…Related inhibitors cover the only five subsite long binding crevice in pancreatic ␣-amylase (8,9,20), and occupy part of the longer binding sites in microbial ␣-amylases (11,13,21) and in cyclodextrin glucosyltransferase (CGTase) (16,22,23). The structures validate modeled substrate complexes and subsite maps (8,12,24,25) by highlighting (i) aromatic stacking and hydrogen bonds between carbohydrate and protein (9,10,21,24,26,27), (ii) conformational features of the bound carbohydrate (8,21,28), (iii) conserved geometry of the catalytic site (10,14,15,21,22,29,30), and (iv) substrate binding motifs in ␤ 3 ␣ loops of the catalytic (␤/␣) 8 barrel (15). The macromolecular substrate starch most probably also interacts with distinct areas outside the cleft as suggested by oligosaccharide occupation at so-called surface or secondary sites in several structures from GH-H (10,14,28,31).…”

Trehalulose Synthase Native and Carbohydrate Complexed Structures Provide Insights into Sucrose Isomerization

“…Besides the narrow size of the pocket, this may explain the weak hydrolytic activity of these enzymes because there is little room for water to enter when a substrate molecule occupies the pocket. In other glycoside hydrolases, optimized systems have been described in which the active site can be continuously and efficiently supplied with water (55)(56)(57). As concerns the water channel connecting the surface to the catalytic pocket, also identified in AS (46), its function is still uncertain.…”