1bsi: Difference between revisions

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[[Image:1bsi.gif|left|200px]]<br />

[[Image:1bsi.gif|left|200px]]<br /><applet load="1bsi" size="350" color="white" frame="true" align="right" spinBox="true"

<applet load="1bsi" size="~~450~~" color="white" frame="true" align="right" spinBox="true"

caption="1bsi, resolution 2.Å" />

'''HUMAN PANCREATIC ALPHA-AMYLASE FROM PICHIA PASTORIS, GLYCOSYLATED PROTEIN'''<br />

==Overview==

Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic, yeast Pichia pastoris and two mutants (D197A and D197N) of a completely, conserved active site carboxylic acid were generated. All recombinant, proteins were shown by electrospray ionization mass spectrometry (ESI-MS), to be glycosylated and the site of attachment was shown to be Asn461 by, peptide mapping in conjunction with ESI-MS. Treatment of these proteins, with endoglycosidase F demonstrated that they contained a single N-linked, oligosaccharide and yielded a protein product with a single N-acetyl, glucosamine (GlcNAc), which could be crystallized. Solution of the crystal, structure to a resolution of 2.0 A confirmed the location of the glycosyl, group as Asn461 and showed that the recombinant protein had essentially, the same conformation as the native enzyme. The kinetic parameters of the, glycosylated and deglycosylated wild-type proteins were the same while the, k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the, wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm, that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.

Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 A confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.

==About this Structure==

1BSI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with NAG, CA and CL as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alpha-amylase Alpha-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.1 3.2.1.1] Full crystallographic information is available from [http://~~ispc~~.weizmann.ac.il/oca-bin/ocashort?id=1BSI OCA].

1BSI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=NAG:'>NAG</scene>, <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=CL:'>CL</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alpha-amylase Alpha-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.1 3.2.1.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1BSI OCA].

==Reference==

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[[Category: Homo sapiens]]

[[Category: Single protein]]

[[Category: Brayer, G.D.]]

[[Category: Brayer, G D.]]

[[Category: Cote, H.C.F.]]

[[Category: Cote, H C.F.]]

[[Category: Hewitt, J.]]

[[Category: Macgillivray, R.T.A.]]

[[Category: Macgillivray, R T.A.]]

[[Category: Numao, S.]]

[[Category: Overall, C.M.]]

[[Category: Overall, C M.]]

[[Category: Rydberg, E.H.]]

[[Category: Rydberg, E H.]]

[[Category: Sidhu, G.]]

[[Category: Vo, H.C.]]

[[Category: Vo, H C.]]

[[Category: Wang, Y.]]

[[Category: Withers, S.G.]]

[[Category: Withers, S G.]]

[[Category: CA]]

[[Category: CL]]

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[[Category: pichia pastoris]]

''Page seeded by [http://~~ispc~~.weizmann.ac.il/oca OCA ] on ~~Mon Nov 12 16~~:14:~~02 2007~~''

''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 11:58:40 2008''

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@@ Line 1: / Line 1: @@
-[[Image:1bsi.gif|left|200px]]<br />
+[[Image:1bsi.gif|left|200px]]<br /><applet load="1bsi" size="350" color="white" frame="true" align="right" spinBox="true"
-<applet load="1bsi" size="450" color="white" frame="true" align="right" spinBox="true"
 caption="1bsi, resolution 2.&Aring;" />
 '''HUMAN PANCREATIC ALPHA-AMYLASE FROM PICHIA PASTORIS, GLYCOSYLATED PROTEIN'''<br />
 ==Overview==
-Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic, yeast Pichia pastoris and two mutants (D197A and D197N) of a completely, conserved active site carboxylic acid were generated. All recombinant, proteins were shown by electrospray ionization mass spectrometry (ESI-MS), to be glycosylated and the site of attachment was shown to be Asn461 by, peptide mapping in conjunction with ESI-MS. Treatment of these proteins, with endoglycosidase F demonstrated that they contained a single N-linked, oligosaccharide and yielded a protein product with a single N-acetyl, glucosamine (GlcNAc), which could be crystallized. Solution of the crystal, structure to a resolution of 2.0 A confirmed the location of the glycosyl, group as Asn461 and showed that the recombinant protein had essentially, the same conformation as the native enzyme. The kinetic parameters of the, glycosylated and deglycosylated wild-type proteins were the same while the, k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the, wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm, that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.
+Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 A confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.
 ==About this Structure==
-BSI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with NAG, CA and CL as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alpha-amylase Alpha-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.1 3.2.1.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1BSI OCA].
+BSI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=NAG:'>NAG</scene>, <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=CL:'>CL</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alpha-amylase Alpha-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.1 3.2.1.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1BSI OCA].
 ==Reference==
@@ Line 15: / Line 14: @@
 [[Category: Homo sapiens]]
 [[Category: Single protein]]
-[[Category: Brayer, G.D.]]
+[[Category: Brayer, G D.]]
-[[Category: Cote, H.C.F.]]
+[[Category: Cote, H C.F.]]
 [[Category: Hewitt, J.]]
-[[Category: Macgillivray, R.T.A.]]
+[[Category: Macgillivray, R T.A.]]
 [[Category: Numao, S.]]
-[[Category: Overall, C.M.]]
+[[Category: Overall, C M.]]
-[[Category: Rydberg, E.H.]]
+[[Category: Rydberg, E H.]]
 [[Category: Sidhu, G.]]
-[[Category: Vo, H.C.]]
+[[Category: Vo, H C.]]
 [[Category: Wang, Y.]]
-[[Category: Withers, S.G.]]
+[[Category: Withers, S G.]]
 [[Category: CA]]
 [[Category: CL]]
@@ Line 40: / Line 39: @@
 [[Category: pichia pastoris]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 12 16:14:02 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 11:58:40 2008''