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

Lindén, Anni‐Maija; Mayans, Olga; Meyer‐Klaucke, Wolfram; Antranikian, Garabed; Wilmanns, Matthias

doi:10.1074/jbc.m211339200

Cited by 102 publications

(91 citation statements)

References 51 publications

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“…Structural studies of ␣-amylases in complex with substrate analogues have received much attention in the past decade (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The pseudotetrasaccharide inhibitor acarbose was used for the vast majority of these studies.…”

mentioning

confidence: 99%

Oligosaccharide Binding to Barley α-Amylase 1

Robert

Haser

Mori

et al. 2005

Journal of Biological Chemistry

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Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley ␣-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley ␣-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely ؊7 through ؉2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley ␣-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1 D180A ) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."

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confidence: 99%

Oligosaccharide Binding to Barley α-Amylase 1

Robert

Haser

Mori

et al. 2005

Journal of Biological Chemistry

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“…Stability of α-amylase activity by Ca 2+ ions at high temperatures has been reported [41]. Enhancement of amylase activity by metallic ions might be due to their ability interact with negatively charged amino acid residues such as aspartic and glutamic acid [42], thereby stabilizing and maintaining the enzyme conformation. Researchers have reported inhibition of amylase activity by Ag + , Hg 2+ , Cd 2+ , Cu 2+ , Pb 2+ , Fe 2+ , Ni 2+ , Mn 2+ and Zn 2+ ions [10,37,43].…”

Section: Characterization Of α-Amylasementioning

confidence: 99%

Purification and Characterization of α-Amylase from Bacillus subtilis Isolated from Cassava Processing Sites

David¹,

Femi²,

Gbenga³

et al. 2017

J Bioremediat Biodegrad

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This study was designed to purify and characterize of α-amylase from pure strain of Bacillus subtilis. The crude α-amylase was purified by ammonium sulphate precipitation, then loaded on DEAE Sephadex A-50 ion exchange chromatography and gel filtration. The effect of pH, temperature and metal ions were investigated on the purified enzyme. The single protein band on SDS-PAGE suggested that the enzyme was homogenous. Two different activity peaks were observed in ion exchange chromatography designated pool A and pool B with the 8% and 4% yield, 15.93 and 6.44 purification fold and specific activity 2.55 μmol/min/mg and 1.03 μmol/min/mg respectively. The two fractions revealed the same optimum pH 7.0 for the α-amylase activity while the enzyme was relatively stable at pH 4.0 and 7.0 between 20 to 40 minutes and 60 to 80 minutes for pool A and pH 8.0 between 40 and 100 minutes for pool B. At 40°C, optimum temperature was reached, and amylase activity was maintained at 75% and 70% temperature stability between 60 to 80 minutes for pool A and B, less than 20%, the residual activity at 60°C and 70°C was recorded. The incubation of α-amylase with Na + and Zn 2+ ions enhanced/activate the enzyme activity correspondingly, Al 3+ and K + ions exhibited varied degree of inhibition while Ca 2+ and Hg 2+ ions caused total inhibition on α-amylase activity. The ability of purified α-amylase from Bacillus subtilis under wide range of temperatures and pH suggests its applications in industries and bioremediation of effluent discharge on food processing sites.

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“…All known -amylases, with a few exceptions, contain a conserved Ca 2+ binding site which is located at the interface between domains A and B (Linden et al, 2003;Prakash & Jaiswal, 2010). In addition, -amylase produced by Bacillus thermooleovorans was found to contain a chloride ion binding site in their active site (Malhotra et al, 2000), which has been shown to enhance the catalytic efficiency of the enzyme, presumably by elevating the pKa of the hydrogen-donating residue in the active site (Prakash & Jaiswal, 2010).…”

Section: Modular Structurementioning

confidence: 99%

“…Most known -amylases, with a few exceptions, contain a conserved Ca 2+ binding site (Linden et al, 2003;Prakash & Jaiswal, 2010) which make calcium be important to the enzyme activity. In manufacture of fructose syrup, the Ca 2+ ions inhibit the glucose isomerase enzyme used in the final step of the process (Tonkova, 2006) and may lead to the formation of inorganic precipitates which have deleterious effects on fermentation and downstream processing (Kelly et al, 2009).…”

Section: Industrial Desirable Aspectsmentioning

confidence: 99%