1lrk: Difference between revisions

Revision as of 14:47, 21 February 2008

Crystal Structure of Escherichia coli UDP-Galactose 4-Epimerase Mutant Y299C Complexed with UDP-N-acetylglucosamine

OverviewOverview

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-Gal and UDP-Glc during normal galactose metabolism. The mammalian form of the enzyme, unlike its Escherichia coli counterpart, can also interconvert UDP-GalNAc and UDP-GlcNAc. One key feature of the epimerase reaction mechanism is the rotation of a 4-ketopyranose intermediate in the active site. By comparing the high resolution x-ray structures of both the bacterial and human forms of the enzyme, it was previously postulated that the additional activity in the human epimerase was due to replacement of the structural equivalent of Tyr-299 in the E. coli enzyme with a cysteine residue, thereby leading to a larger active site volume. To test this hypothesis, the Y299C mutant form of the E. coli enzyme was prepared and its three-dimensional structure solved as described here. Additionally, the Y299C mutant protein was assayed for activity against both UDP-Gal and UDP-GalNAc. These studies have revealed that, indeed, this simple mutation did confer UDP-GalNAc/UDP-GlcNAc converting activity to the bacterial enzyme with minimal changes in its three-dimensional structure. Specifically, although the Y299C mutation in the bacterial enzyme resulted in a loss of epimerase activity with regard to UDP-Gal by almost 5-fold, it resulted in a gain of activity against UDP-GalNAc by more than 230-fold.

About this StructureAbout this Structure

1LRK is a Single protein structure of sequence from Escherichia coli with , , and as ligands. Active as UDP-glucose 4-epimerase, with EC number 5.1.3.2 Full crystallographic information is available from OCA.

ReferenceReference

Structural analysis of the Y299C mutant of Escherichia coli UDP-galactose 4-epimerase. Teaching an old dog new tricks., Thoden JB, Henderson JM, Fridovich-Keil JL, Holden HM, J Biol Chem. 2002 Jul 26;277(30):27528-34. Epub 2002 May 17. PMID:12019271

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-[[Image:1lrk.jpg|left|200px]]<br /><applet load="1lrk" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1lrk.jpg|left|200px]]<br /><applet load="1lrk" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1lrk, resolution 1.75&Aring;" />
 '''Crystal Structure of Escherichia coli UDP-Galactose 4-Epimerase Mutant Y299C Complexed with UDP-N-acetylglucosamine'''<br />
 ==Overview==
-UDP-galactose 4-epimerase catalyzes the interconversion of UDP-Gal and, UDP-Glc during normal galactose metabolism. The mammalian form of the, enzyme, unlike its Escherichia coli counterpart, can also interconvert, UDP-GalNAc and UDP-GlcNAc. One key feature of the epimerase reaction, mechanism is the rotation of a 4-ketopyranose intermediate in the active, site. By comparing the high resolution x-ray structures of both the, bacterial and human forms of the enzyme, it was previously postulated that, the additional activity in the human epimerase was due to replacement of, the structural equivalent of Tyr-299 in the E. coli enzyme with a cysteine, residue, thereby leading to a larger active site volume. To test this, hypothesis, the Y299C mutant form of the E. coli enzyme was prepared and, its three-dimensional structure solved as described here. Additionally, the Y299C mutant protein was assayed for activity against both UDP-Gal and, UDP-GalNAc. These studies have revealed that, indeed, this simple mutation, did confer UDP-GalNAc/UDP-GlcNAc converting activity to the bacterial, enzyme with minimal changes in its three-dimensional structure., Specifically, although the Y299C mutation in the bacterial enzyme resulted, in a loss of epimerase activity with regard to UDP-Gal by almost 5-fold, it resulted in a gain of activity against UDP-GalNAc by more than, 230-fold.
+UDP-galactose 4-epimerase catalyzes the interconversion of UDP-Gal and UDP-Glc during normal galactose metabolism. The mammalian form of the enzyme, unlike its Escherichia coli counterpart, can also interconvert UDP-GalNAc and UDP-GlcNAc. One key feature of the epimerase reaction mechanism is the rotation of a 4-ketopyranose intermediate in the active site. By comparing the high resolution x-ray structures of both the bacterial and human forms of the enzyme, it was previously postulated that the additional activity in the human epimerase was due to replacement of the structural equivalent of Tyr-299 in the E. coli enzyme with a cysteine residue, thereby leading to a larger active site volume. To test this hypothesis, the Y299C mutant form of the E. coli enzyme was prepared and its three-dimensional structure solved as described here. Additionally, the Y299C mutant protein was assayed for activity against both UDP-Gal and UDP-GalNAc. These studies have revealed that, indeed, this simple mutation did confer UDP-GalNAc/UDP-GlcNAc converting activity to the bacterial enzyme with minimal changes in its three-dimensional structure. Specifically, although the Y299C mutation in the bacterial enzyme resulted in a loss of epimerase activity with regard to UDP-Gal by almost 5-fold, it resulted in a gain of activity against UDP-GalNAc by more than 230-fold.
 ==About this Structure==
-LRK is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with NA, NAD, UD1 and PGE as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/UDP-glucose_4-epimerase UDP-glucose 4-epimerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.1.3.2 5.1.3.2] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1LRK OCA].
+LRK is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=NA:'>NA</scene>, <scene name='pdbligand=NAD:'>NAD</scene>, <scene name='pdbligand=UD1:'>UD1</scene> and <scene name='pdbligand=PGE:'>PGE</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/UDP-glucose_4-epimerase UDP-glucose 4-epimerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.1.3.2 5.1.3.2] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1LRK OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Single protein]]
 [[Category: UDP-glucose 4-epimerase]]
-[[Category: Fridovich-Keil, J.L.]]
+[[Category: Fridovich-Keil, J L.]]
-[[Category: Henderson, J.M.]]
+[[Category: Henderson, J M.]]
-[[Category: Holden, H.M.]]
+[[Category: Holden, H M.]]
-[[Category: Thoden, J.B.]]
+[[Category: Thoden, J B.]]
 [[Category: NA]]
 [[Category: NAD]]
@@ Line 26: / Line 26: @@
 [[Category: short chain dehydrogenase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 20:48:45 2007''
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