Literature summary for 3.2.1.1 extracted from

Tan, T.C.; Mijts, B.N.; Swaminathan, K.; Patel, B.K.; Divne, C.
Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N-domain with a role in binding raw starch (2008), J. Mol. Biol., 378, 852-870.

Cloned(Commentary)

Cloned (Comment)	Organism
expressed ion Escherichia coli BL21(DE3)	Halothermothrix orenii

Crystallization (Commentary)

Crystallization (Comment)	Organism
two complexes of AmyB with carbohydrate ligands: the 1.35-A-resolution structure with the acarbose transglycosylation product, and the 2.2-A-resolution conplex of AmyB with alpha-cyclodextrin and hydrolysis products of maltoheptaose. The AmyB-acarbose complex includes residues 15-599, 1 acarbose molecule, 1 acarbose derived nonsaccharide, 2 glucose molecules, 4 calcium ions, 1 sodium ion, and 576 water molecules. The AmyB-maltoheptaose/cyclodextrin complex includes residues 14-599, 2 maltotetraose molecules, 1 alpha-cyclodextrin molecule, 7 calcium ions, 1 sodium ion, and 260 water molecules. The active site in AMyB is located at the C-terminal end of TIM barrel, with the residues Asp350, Glu380, and Asp447 as the catalytic residues	Halothermothrix orenii

Crystallization (Comment)

Organism

two complexes of AmyB with carbohydrate ligands: the 1.35-A-resolution structure with the acarbose transglycosylation product, and the 2.2-A-resolution conplex of AmyB with alpha-cyclodextrin and hydrolysis products of maltoheptaose. The AmyB-acarbose complex includes residues 15-599, 1 acarbose molecule, 1 acarbose derived nonsaccharide, 2 glucose molecules, 4 calcium ions, 1 sodium ion, and 576 water molecules. The AmyB-maltoheptaose/cyclodextrin complex includes residues 14-599, 2 maltotetraose molecules, 1 alpha-cyclodextrin molecule, 7 calcium ions, 1 sodium ion, and 260 water molecules. The active site in AMyB is located at the C-terminal end of TIM barrel, with the residues Asp350, Glu380, and Asp447 as the catalytic residues

Halothermothrix orenii

Protein Variants

Protein Variants	Comment	Organism
DELTAAmyB	lacking the N-domain, with no significant difference between the rates of soluble starch degradation, indicating that the N-domain does not play a direct role in catalysis with this substrate. For insoluble starch AmyB shows increase binding compared with DELTAAmyB, suggesting that the N-domain enhances the ability of AmyB to bind this substrate. The temperature stability of AmyB and DELTAAmyB, lacking the N-domain are strongly influenced by NaCl concentration, shown by an increasing melting temperature with increased NaCl concentrations up to 4-4.5 M	Halothermothrix orenii

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
membrane	bound	Halothermothrix orenii	16020	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	strictly dependent, retaining below 20% activity in the absence of CaCl2. The optimal concentration is ca. 0.2 mM	Halothermothrix orenii
NaCl	the enzyme is active over a broad range of salt concentrations, with optimum activity at 0.9 M. At 1.7, 2.6, and 4.3 M NaCl AmyB ist 80, 60, and 12% active, respectively. AmyB is a halophilic enzyme, but is still above 45% active in the absence of salt	Halothermothrix orenii

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
71000	-	excluding the lipoprotein signal peptide	Halothermothrix orenii

Organism

Organism	UniProt	Comment	Textmining
Halothermothrix orenii	-	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
alpha-cyclodextrin + H2O	1.1% relative activity compared to amylose as substrate, pH 8.0. 65°C	Halothermothrix orenii	?	-	?
amylopectin + H2O	74.6% relative activity compared to amylose as substrate, pH 8.0. 65°C	Halothermothrix orenii	?	-	?
amylose + H2O	100% relative activity as reference for substrate specificity studies, pH 8.0. 65°C	Halothermothrix orenii	?	-	?
beta-cyclodextrin + H2O	3.7% relative activity compared to amylose as substrate, pH 8.0. 65°C	Halothermothrix orenii	?	-	?
gamma-cyclodextrin + H2O	2.6% relative activity compared to amylose as substrate, pH 8.0. 65°C	Halothermothrix orenii	?	-	?
glycogen + H2O	16.7% relative activity compared to amylose as substrate, pH 8.0. 65°C	Halothermothrix orenii	?	-	?
pullulan + H2O	2.5% relative activity compared to amylose as substrate, pH 8.0. 65°C	Halothermothrix orenii	?	-	?
starch + H2O	soluble starch, 93.9% relative activity compared to amylose as substrate, pH 8.0, 65°C	Halothermothrix orenii	?	-	?

Synonyms

Synonyms	Comment	Organism
AmyB	-	Halothermothrix orenii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
65	-	optimum and assay at	Halothermothrix orenii

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
37	80	retains above 40% activity within the range of 37-80°C, with an optimum at 65°C	Halothermothrix orenii

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
additional information	-	the temperature stability of AmyB and DELTAAmyB, lacking the N-domain are strongly influenced by NaCl concentration, shown by an increasing melting temperature with increased NaCl concentrations up to 4-4.5 M	Halothermothrix orenii
60	-	fully active after preincubation at 60°C in the presence of soluble starch. In the absence of starch AmyB is inactivated rapidly after 15 min of preincubation	Halothermothrix orenii
63	-	Tm value, the maximum thermal stability for AmyB and DELTAAmyB, lacking the N-domain are obtained at pH above 6.0 and above 3.0 M NaCl	Halothermothrix orenii
80	-	after preincubation in the presence of starch, activity falls off linearly with time to reach 10% after 2 h, with a half-life of above 1 h at 80°C. In the absence of starch AmyB is inactivated rapidly after 15 min of preincubation	Halothermothrix orenii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	-	Halothermothrix orenii
8	-	assay at	Halothermothrix orenii

pH Range

pH Minimum	pH Maximum	Comment	Organism
5.2	9.3	above 20% activity within the range of pH 5.2-9.3, with an optimum at pH 7.0	Halothermothrix orenii

pH Stability

pH Stability	pH Stability Maximum	Comment	Organism
5	-	AmyB and the variant DELTAAmyB, lacking the N-domain depend only weakly on pH. At pH 5.0 both proteins are destabilized	Halothermothrix orenii
7	-	AmyB and the variant DELTAAmyB, lacking the N-domain depend only weakly on pH. At pH 7.0 both proteins are equally stabilized	Halothermothrix orenii
7.5	8.5	AmyB and the variant DELTAAmyB, lacking the N-domain depend only weakly on pH. At 7.5-8.5 they show almost identical Tm values	Halothermothrix orenii

pI Value

Organism	Comment	pI Value Maximum	pI Value
Halothermothrix orenii	theoretical value	-	4.4