1tw5
From Proteopedia
(New page: 200px<br /><applet load="1tw5" size="450" color="white" frame="true" align="right" spinBox="true" caption="1tw5, resolution 2.30Å" /> '''beta1,4-galactosyltr...) |
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| - | [[Image:1tw5.gif|left|200px]]<br /><applet load="1tw5" size=" | + | [[Image:1tw5.gif|left|200px]]<br /><applet load="1tw5" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1tw5, resolution 2.30Å" /> | caption="1tw5, resolution 2.30Å" /> | ||
'''beta1,4-galactosyltransferase mutant M344H-Gal-T1 in complex with Chitobiose'''<br /> | '''beta1,4-galactosyltransferase mutant M344H-Gal-T1 in complex with Chitobiose'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Beta-1,4-galactosyltransferase (beta4Gal-T1) in the presence of manganese | + | Beta-1,4-galactosyltransferase (beta4Gal-T1) in the presence of manganese ion transfers galactose from UDP-galactose (UDP-Gal) to N-acetylglucosamine (GlcNAc) that is either free or linked to an oligosaccharide. Crystallographic studies on bovine beta4Gal-T1 have shown that the primary metal binding site is located in the hinge region of a long flexible loop, which upon Mn(2+) and UDP-Gal binding changes from an open to a closed conformation. This conformational change creates an oligosaccharide binding site in the enzyme. Neither UDP nor UDP analogues efficiently induce these conformational changes in the wild-type enzyme, thereby restricting the structural analysis of the acceptor binding site. The binding of Mn(2+) involves an uncommon coordination to the Sdelta atom of Met344; when it is mutated to His, the mutant M344H, in the presence of Mn(2+) and UDP-hexanolamine, readily changes to a closed conformation, facilitating the structural analysis of the enzyme bound with an oligosaccharide acceptor. Although the mutant M344H loses 98% of its Mn(2+)-dependent activity, it exhibits 25% of its activity in the presence of Mg(2+). The crystal structures of M344H-Gal-T1 in complex with either UDP-Gal.Mn(2+) or UDP-Gal.Mg(2+), determined at 2.3 A resolution, show that the mutant enzyme in these complexes is in a closed conformation, and the coordination stereochemistry of Mg(2+) is quite similar to that of Mn(2+). Although either Mn(2+) or Mg(2+), together with UDP-Gal, binds and changes the conformation of the M344H mutant to the closed one, it is the Mg(2+) complex that engages efficiently in catalyses. Thus, this property enabled us to crystallize the M344H mutant for the first time with the acceptor substrate chitobiose in the presence of UDP-hexanolamine and Mn(2+). The crystal structure determined at 2.3 A resolution reveals that the GlcNAc residue at the nonreducing end of chitobiose makes extensive hydrophobic interactions with the highly conserved Tyr286 residue. |
==About this Structure== | ==About this Structure== | ||
| - | 1TW5 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with CBS, MN, SO4, UDH, MES and DIO as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http:// | + | 1TW5 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with <scene name='pdbligand=CBS:'>CBS</scene>, <scene name='pdbligand=MN:'>MN</scene>, <scene name='pdbligand=SO4:'>SO4</scene>, <scene name='pdbligand=UDH:'>UDH</scene>, <scene name='pdbligand=MES:'>MES</scene> and <scene name='pdbligand=DIO:'>DIO</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1TW5 OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Boeggeman, E.]] | [[Category: Boeggeman, E.]] | ||
| - | [[Category: Qasba, P | + | [[Category: Qasba, P K.]] |
[[Category: Ramakrishnan, B.]] | [[Category: Ramakrishnan, B.]] | ||
[[Category: CBS]] | [[Category: CBS]] | ||
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[[Category: met344his mutation; closed conformation; chitobiose binding]] | [[Category: met344his mutation; closed conformation; chitobiose binding]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:18:10 2008'' |
Revision as of 13:18, 21 February 2008
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beta1,4-galactosyltransferase mutant M344H-Gal-T1 in complex with Chitobiose
Overview
Beta-1,4-galactosyltransferase (beta4Gal-T1) in the presence of manganese ion transfers galactose from UDP-galactose (UDP-Gal) to N-acetylglucosamine (GlcNAc) that is either free or linked to an oligosaccharide. Crystallographic studies on bovine beta4Gal-T1 have shown that the primary metal binding site is located in the hinge region of a long flexible loop, which upon Mn(2+) and UDP-Gal binding changes from an open to a closed conformation. This conformational change creates an oligosaccharide binding site in the enzyme. Neither UDP nor UDP analogues efficiently induce these conformational changes in the wild-type enzyme, thereby restricting the structural analysis of the acceptor binding site. The binding of Mn(2+) involves an uncommon coordination to the Sdelta atom of Met344; when it is mutated to His, the mutant M344H, in the presence of Mn(2+) and UDP-hexanolamine, readily changes to a closed conformation, facilitating the structural analysis of the enzyme bound with an oligosaccharide acceptor. Although the mutant M344H loses 98% of its Mn(2+)-dependent activity, it exhibits 25% of its activity in the presence of Mg(2+). The crystal structures of M344H-Gal-T1 in complex with either UDP-Gal.Mn(2+) or UDP-Gal.Mg(2+), determined at 2.3 A resolution, show that the mutant enzyme in these complexes is in a closed conformation, and the coordination stereochemistry of Mg(2+) is quite similar to that of Mn(2+). Although either Mn(2+) or Mg(2+), together with UDP-Gal, binds and changes the conformation of the M344H mutant to the closed one, it is the Mg(2+) complex that engages efficiently in catalyses. Thus, this property enabled us to crystallize the M344H mutant for the first time with the acceptor substrate chitobiose in the presence of UDP-hexanolamine and Mn(2+). The crystal structure determined at 2.3 A resolution reveals that the GlcNAc residue at the nonreducing end of chitobiose makes extensive hydrophobic interactions with the highly conserved Tyr286 residue.
About this Structure
1TW5 is a Single protein structure of sequence from Bos taurus with , , , , and as ligands. Full crystallographic information is available from OCA.
Reference
Effect of the Met344His mutation on the conformational dynamics of bovine beta-1,4-galactosyltransferase: crystal structure of the Met344His mutant in complex with chitobiose., Ramakrishnan B, Boeggeman E, Qasba PK, Biochemistry. 2004 Oct 5;43(39):12513-22. PMID:15449940
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