Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis.


Abstract

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of DeltaHcal values after consecutive calorimetric transitions, a DeltaHcal/DeltaHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7 degrees C, a DeltaHcal of 238 kcal mol-1, and a DeltaCp of 8.47 kcal mol-1 K-1. These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a) the specific DeltaGmax of AHA [22 cal (mol of residue)-1] is 4 times lower than that of mesophilic alpha-amylases, (b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state. Study holds ProTherm entries: 5645, 5646, 5647, 5648, 5649, 5650, 5651, 5652, 5653, 5654, 5655, 5656, 5657, 5658, 5659, 5660, 5661, 5662, 5663, 5664, 5665, 5666, 5667 Extra Details:

Submission Details

ID: mYBtMV993

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details
Feller G;d'Amico D;Gerday C,Biochemistry (1999) Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis. PMID:10194383
Additional Information

Study Summary

Number of data points 55
Proteins Pancreatic alpha-amylase ; GLYCOSYLTRANSFERASE ; Alpha-amylase ; Alpha-amylase ; Alpha-amylase 1 ; Alpha-amylase 1 ; Alpha-amylase ; Alpha-amylase ; Alpha-amylase
Unique complexes 6
Assays/Quantities/Protocols Experimental Assay: dHvH pH:7.2, buffers:MOPS: 30 mM ; Experimental Assay: dHcal buffers:MES: 30 mM, pH:6.4 ; Experimental Assay: Tm buffers:MES: 30 mM, pH:6.4 ; Experimental Assay: dHvH buffers:MES: 30 mM, pH:6.4 ; Experimental Assay: dHcal buffers:MES: 30 mM, pH:6.3 ; Experimental Assay: Tm buffers:MES: 30 mM, pH:6.3 ; Experimental Assay: dHvH buffers:MES: 30 mM, pH:6.3 ; Experimental Assay: dHcal pH:6.2, buffers:MES: 30 mM ; Experimental Assay: Tm pH:6.2, buffers:MES: 30 mM ; Experimental Assay: dHvH pH:6.2, buffers:MES: 30 mM ; Experimental Assay: dHcal buffers:MES: 30 mM, pH:6.1 ; Experimental Assay: Tm buffers:MES: 30 mM, pH:6.1 ; Experimental Assay: dHvH buffers:MES: 30 mM, pH:6.1 ; Experimental Assay: dHcal pH:6.0, buffers:MES: 30 mM ; Experimental Assay: Tm pH:6.0, buffers:MES: 30 mM ; Experimental Assay: dHvH pH:6.0, buffers:MES: 30 mM ; Experimental Assay: dHcal buffers:MES: 30 mM, pH:5.9 ; Experimental Assay: Tm buffers:MES: 30 mM, pH:5.9 ; Experimental Assay: dHvH buffers:MES: 30 mM, pH:5.9 ; Experimental Assay: dHcal pH:5.8, buffers:MES: 30 mM ; Experimental Assay: Tm pH:5.8, buffers:MES: 30 mM ; Experimental Assay: dHvH pH:5.8, buffers:MES: 30 mM ; Experimental Assay: dHcal pH:5.7, buffers:MES: 30 mM ; Experimental Assay: Tm pH:5.7, buffers:MES: 30 mM ; Experimental Assay: dHvH pH:5.7, buffers:MES: 30 mM ; Experimental Assay: dHcal pH:7.2, buffers:MOPS: 30 mM ; Experimental Assay: Tm pH:7.2, buffers:MOPS: 30 mM
Libraries Mutations for sequence QYAPQTQSGRTSIVHLFEWRWVDIALECERYLGPKGFGGVQVSPPNENVVVTNPSRPWWERYQPVSYKLCTRSGNENEFRDMVTRCNNVGVRIYVDAVINHMCGSGAAAGTGTTCGSYCNPGSREFPAVPYSAWDFNDGKCKTASGGIESYNDPYQVRDCQLVGLLDLALEKDYVRSMIADYLNKLIDIGVAGFRIDASKHMWPGDIKAVLDKLHNLNTNWFPAGSRPFIFQEVIDLGGEAIKSSEYFGNGRVTEFKYGAKLGTVVRKWSGEKMSYLKNWGEGWGFMPSDRALVFVDNHDNQRGHGAGGSSILTFWDARLYKVAVGFMLAHPYGFTRVMSSYRWARNFVNGEDVNDWIGPPNNNGVIKEVTINADTTCGNDWVCEHRWREIRNMVWFRNVVDGEPFANWWDNGSNQVAFGRGNRGFIVFNNDDWQLSSTLQTGLPGGTYCDVISGDKVGNSCTGIKVYVSSDGTAQFSISNSAEDPFIAIHAESKL ; Mutations for sequence TPTTFVHLFEWNWQDVAQECEQYLGPKGYAAVQVSPPNEHITGSQWWTRYQPVSYELQSRGGNRAQFIDMVNRCSAAGVDIYVDTLINHMAAGSGTGTAGNSFGNKSFPIYSPQDFHESCTINNSDYGNDRYRVQNCELVGLADLDTASNYVQNTIAAYINDLQAIGVKGFRFDASKHVAASDIQSLMAKVNGSPVVFQEVIDQGGEAVGASEYLSTGLVTEFKYSTELGNTFRNGSLAWLSNFGEGWGFMPSSSAVVFVDNHDNQRGHGGAGNVITFEDGRLYDLANVFMLAYPYGYPKVMSSYDFHGDTDAGGPNVPVHNNGNLECFASNWKCEHRWSYIAGGVDFRNNTADNWAVTNWWDNTNNQISFGRGSSGHMAINKEDSTLTATVQTDMASGQYCNVLKGELSADAKSCSGEVITVNSDGTINLNIGAWDAMAIHKNAKLNTSSAS ; Mutations for sequence MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAVNGTLMQYFEWYTPNDGQHWKRLQNDAEHLSDIGITAVWIPPAYKGLSQSDNGYGPYDLYDLGEFQQKGTVRTKYGTKSELQDAIGSLHSRNVQVYGDVVLNHKAGADATEDVTAVEVNPANRNQETSEEYQIKAWTDFRFPGRGNTYSDFKWHWYHFDGADWDESRKISRIFKFRGEGKAWDWEVSSENGNYDYLMYADVDYDHPDVVAETKKWGIWYANELSLDGFRIDAAKHIKFSFLRDWVQAVRQATGKEMFTVAEYWQNNAGKLENYLNKTSFNQSVFDVPLHFNLQAASSQGGGYDMRRLLDGTVVSRHPEKAVTFVENHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGTSPKEIPSLKDNIEPILKARKEYAYGPQHDYIDHPDVIGWTREGDSSAAKSGLAALITDGPGGSKRMYAGLKNAGETWYDITGNRSDTVKIGSDGWGEFHVNDGSVSIYVQK ; Mutations for sequence EYSSNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIHNPFRPWWERYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFNPGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLSGLLDLALGKDYVRSKIAEYMNHLIDIGVAGFRIDASKHMWPGDIKAILDKLHNLNSNWFPEGSKPFIYQEVIDLGGEPIKSSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFMPSDRALVFVDNHDNQRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRYFENGKDVNDWVGPPNDNGVTKEVTINPDTTCGNDWVCEHRWRQIRNMVNFRNVVDGQPFTNWYDNGSNQVAFGRGNRGFIVFNNDDWTFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSISNSAEDPFIAIHAESKL ; Mutations for sequence A:ANLNGTLMQYFEWYMPNDGQHWKRLQNDSAYLAEHGITAVWIPPAYKGTSQADVGYGAYDLYDLGEFHQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKGGADATEDVTAVEVDPADRNRVISGEHLIKAWTHFHFPGRGSTYSDFKWHWYHFDGTDWDESRKLNRIYKFQGKAWDWEVSNE/B:NGNYDYLMYADIDYDHPDVAAEIKRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVAEYWQNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNSTVVSKHPLKAVTFVDNHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKARKQYAYGAQHDYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYVGRQNAGETWHDITGNRSEPVVINSEGWGEFHVNGGSVSIYVQR ; Mutations for sequence EKDANFASGRNSIVHLFEWKWNDIADECERFLQPQGFGGVQISPPNEYLVADGRPWWERYQPVSYIINTRSGDESAFTDMTRRCNDAGVRIYVDAVINHMTGMNGVGTSGSSADHDGMNYPAVPYGSGDFHSPCEVNNYQDADNVRNCELVGLRDLNQGSDYVRGVLIDYMNHMIDLGVAGFRVDAAKHMSPGDLSVIFSGLKNLNTDYGFADGARPFIYQEVIDLGGEAISKNEYTGFGCVLEFQFGVSLGNAFQGGNQLKNLANWGPEWGLLEGLDAVVFVDNHDNQRTGGSQILTYKNPKPYKMAIAFMLAHPYGTTRIMSSFDFTDNDQGPPQDGSGNLISPGINDDNTCSNGYVCEHRWRQVYGMVGFRNAVEGTQVENWWSNDDNQIAFSRGSQGFVAFTNGGDLNQNLNTGLPAGTYCDVISGELSGGSCTGKSVTVGDNGSADISLGSAEDDGVLAIHVNAKL

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1CLV 1999-05-04T00:00:00+0000 2.0 YELLOW MEAL WORM ALPHA-AMYLASE IN COMPLEX WITH THE AMARANTH ALPHA-AMYLASE INHIBITOR
1JAE 1997-09-30T00:00:00+0000 1.65 STRUCTURE OF TENEBRIO MOLITOR LARVAL ALPHA-AMYLASE
1TMQ 1998-01-13T00:00:00+0000 2.5 STRUCTURE OF TENEBRIO MOLITOR LARVAL ALPHA-AMYLASE IN COMPLEX WITH RAGI BIFUNCTIONAL INHIBITOR
1VIW 1998-07-21T00:00:00+0000 3.0 TENEBRIO MOLITOR ALPHA-AMYLASE-INHIBITOR COMPLEX
1BLI 1998-01-07T00:00:00+0000 1.9 BACILLUS LICHENIFORMIS ALPHA-AMYLASE
1BPL 1995-07-13T00:00:00+0000 2.2 GLYCOSYLTRANSFERASE
1BPL 1995-07-13T00:00:00+0000 2.2 GLYCOSYLTRANSFERASE
1E3X 2000-06-26T00:00:00+0000 1.9 Native structure of chimaeric amylase from B. amyloliquefaciens and B. licheniformis at 1.92A
1E3Z 2000-06-27T00:00:00+0000 1.93 Acarbose complex of chimaeric amylase from B. amyloliquefaciens and B. licheniformis at 1.93A
1E40 2000-06-27T00:00:00+0000 2.2 Tris/maltotriose complex of chimaeric amylase from B. amyloliquefaciens and B. licheniformis at 2.2A

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
98.4 Pancreatic alpha-amylase P00690 AMYP_PIG
197.4 A,B GLYCOSYLTRANSFERASE P06278 AMY_BACLI
100.0 Alpha-amylase P56634 AMY_TENMO
100.0 Alpha-amylase P00692 AMY_BACAM
97.0 Alpha-amylase 1 P04746 AMYP_HUMAN
97.6 Alpha-amylase 1 P19961 AMY2B_HUMAN
100.0 Alpha-amylase 1 P04745 AMY1_HUMAN
100.0 Alpha-amylase P29957 AMY_PSEHA