1lrn: Difference between revisions

Revision as of 14:47, 21 February 2008

Aquifex aeolicus KDO8P synthase H185G mutant in complex with Cadmium

OverviewOverview

Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) by favoring the activation of a water molecule coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233 are the other metal ligands. Wild-type KDO8PS is purified with Zn(2+) or Fe(2+) in the active site, but maximal activity in vitro is achieved when the endogenous metal is replaced with Cd(2+). The H185G enzyme retains 8% of the wild-type activity. ICP mass spectrometry analysis indicates that loss of His185 decreases the enzyme affinity for Fe(2+), but not for Zn(2+). However, maximal activity is again achieved by substitution of the endogenous metal with Cd(2+). We have determined the X-ray structures of the Cd(2+) H185G enzyme in its substrate-free form, and in complex with PEP, and PEP plus A5P. These structures show a normal amount of Cd(2+) bound, suggesting that coordination by His185 is not essential to retain Cd(2+) in the active site. Nonetheless, there are significant changes in the coordination sphere of Cd(2+) with respect to the wild-type enzyme, as the carboxylate moiety of PEP binds directly to the metal ion and replaces water and His185 as ligands. These observations indicate that the primary function of His185 in A.aeolicus KDO8PS is to orient PEP in the active site of the enzyme in such a way that a water molecule on the sinister (si) side of PEP can be activated by direct coordination to the metal ion.

About this StructureAbout this Structure

1LRN is a Single protein structure of sequence from Aquifex aeolicus with and as ligands. Active as 3-deoxy-8-phosphooctulonate synthase, with EC number 2.5.1.55 Full crystallographic information is available from OCA.

ReferenceReference

Function of His185 in Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase., Wang J, Duewel HS, Stuckey JA, Woodard RW, Gatti DL, J Mol Biol. 2002 Nov 22;324(2):205-14. PMID:12441100

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OCA

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-[[Image:1lrn.jpg|left|200px]]<br /><applet load="1lrn" size="450" color="white" frame="true" align="right" spinBox="true"
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 caption="1lrn, resolution 2.10&Aring;" />
 '''Aquifex aeolicus KDO8P synthase H185G mutant in complex with Cadmium'''<br />
 ==Overview==
-Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase, (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and, phosphoenolpyruvate (PEP) by favoring the activation of a water molecule, coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233, are the other metal ligands. Wild-type KDO8PS is purified with Zn(2+) or, Fe(2+) in the active site, but maximal activity in vitro is achieved when, the endogenous metal is replaced with Cd(2+). The H185G enzyme retains 8%, of the wild-type activity. ICP mass spectrometry analysis indicates that, loss of His185 decreases the enzyme affinity for Fe(2+), but not for, Zn(2+). However, maximal activity is again achieved by substitution of the, endogenous metal with Cd(2+). We have determined the X-ray structures of, the Cd(2+) H185G enzyme in its substrate-free form, and in complex with, PEP, and PEP plus A5P. These structures show a normal amount of Cd(2+), bound, suggesting that coordination by His185 is not essential to retain, Cd(2+) in the active site. Nonetheless, there are significant changes in, the coordination sphere of Cd(2+) with respect to the wild-type enzyme, as, the carboxylate moiety of PEP binds directly to the metal ion and replaces, water and His185 as ligands. These observations indicate that the primary, function of His185 in A.aeolicus KDO8PS is to orient PEP in the active, site of the enzyme in such a way that a water molecule on the sinister, (si) side of PEP can be activated by direct coordination to the metal ion.
+Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) by favoring the activation of a water molecule coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233 are the other metal ligands. Wild-type KDO8PS is purified with Zn(2+) or Fe(2+) in the active site, but maximal activity in vitro is achieved when the endogenous metal is replaced with Cd(2+). The H185G enzyme retains 8% of the wild-type activity. ICP mass spectrometry analysis indicates that loss of His185 decreases the enzyme affinity for Fe(2+), but not for Zn(2+). However, maximal activity is again achieved by substitution of the endogenous metal with Cd(2+). We have determined the X-ray structures of the Cd(2+) H185G enzyme in its substrate-free form, and in complex with PEP, and PEP plus A5P. These structures show a normal amount of Cd(2+) bound, suggesting that coordination by His185 is not essential to retain Cd(2+) in the active site. Nonetheless, there are significant changes in the coordination sphere of Cd(2+) with respect to the wild-type enzyme, as the carboxylate moiety of PEP binds directly to the metal ion and replaces water and His185 as ligands. These observations indicate that the primary function of His185 in A.aeolicus KDO8PS is to orient PEP in the active site of the enzyme in such a way that a water molecule on the sinister (si) side of PEP can be activated by direct coordination to the metal ion.
 ==About this Structure==
-LRN is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Aquifex_aeolicus Aquifex aeolicus] with PO4 and CD as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/3-deoxy-8-phosphooctulonate_synthase 3-deoxy-8-phosphooctulonate synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.5.1.55 2.5.1.55] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1LRN OCA].
+LRN is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Aquifex_aeolicus Aquifex aeolicus] with <scene name='pdbligand=PO4:'>PO4</scene> and <scene name='pdbligand=CD:'>CD</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/3-deoxy-8-phosphooctulonate_synthase 3-deoxy-8-phosphooctulonate synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.5.1.55 2.5.1.55] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1LRN OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Aquifex aeolicus]]
 [[Category: Single protein]]
-[[Category: Duewel, H.S.]]
+[[Category: Duewel, H S.]]
-[[Category: Gatti, D.L.]]
+[[Category: Gatti, D L.]]
-[[Category: Stuckey, J.A.]]
+[[Category: Stuckey, J A.]]
 [[Category: Wang, J.]]
-[[Category: Woodard, R.W.]]
+[[Category: Woodard, R W.]]
 [[Category: CD]]
 [[Category: PO4]]
@@ Line 25: / Line 25: @@
 [[Category: kdo8ps]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 20:48:56 2007''
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