1ll6: Difference between revisions

Revision as of 14:46, 21 February 2008

STRUCTURE OF THE D169N MUTANT OF C. IMMITIS CHITINASE 1

OverviewOverview

Allosamidin is a known inhibitor of class 18 chitinases. We show that allosamidin is a competitive inhibitor of the fungal chitinase CiX1 from Coccidioides immitis, with a K(i) of 60 nM. We report the X-ray structure of the complex and show that upon inhibitor binding the side-chain of Asp169 rotates to form an ion pair with the oxazolinium cation. The mechanism of action is thought to involve protonation of the leaving group by Glu171 and substrate assistance by the sugar acetamido moiety to form an oxazoline-like intermediate. We converted both amino acid residues to the corresponding amide and found that each mutation effectively abolishes enzyme activity. X-ray structures show the mutant enzymes retain the basic wild-type structure and that the loss of mutant activity is due to their altered chemical properties. The high affinity of allosamidin, and its similarity to the putative reaction intermediate, suggests it is a transition state analog. This helps validate our contention that the role of Asp169 is to electrostatically stabilize the reaction transition state.

About this StructureAbout this Structure

1LL6 is a Single protein structure of sequence from Coccidioides immitis. Active as Chitinase, with EC number 3.2.1.14 Full crystallographic information is available from OCA.

ReferenceReference

The structure of an allosamidin complex with the Coccidioides immitis chitinase defines a role for a second acid residue in substrate-assisted mechanism., Bortone K, Monzingo AF, Ernst S, Robertus JD, J Mol Biol. 2002 Jul 5;320(2):293-302. PMID:12079386

Page seeded by OCA on Thu Feb 21 13:45:58 2008

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA

@@ Line 1: / Line 1: @@
-[[Image:1ll6.gif|left|200px]]<br /><applet load="1ll6" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1ll6.gif|left|200px]]<br /><applet load="1ll6" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1ll6, resolution 2.8&Aring;" />
 '''STRUCTURE OF THE D169N MUTANT OF C. IMMITIS CHITINASE 1'''<br />
 ==Overview==
-Allosamidin is a known inhibitor of class 18 chitinases. We show that, allosamidin is a competitive inhibitor of the fungal chitinase CiX1 from, Coccidioides immitis, with a K(i) of 60 nM. We report the X-ray structure, of the complex and show that upon inhibitor binding the side-chain of, Asp169 rotates to form an ion pair with the oxazolinium cation. The, mechanism of action is thought to involve protonation of the leaving group, by Glu171 and substrate assistance by the sugar acetamido moiety to form, an oxazoline-like intermediate. We converted both amino acid residues to, the corresponding amide and found that each mutation effectively abolishes, enzyme activity. X-ray structures show the mutant enzymes retain the basic, wild-type structure and that the loss of mutant activity is due to their, altered chemical properties. The high affinity of allosamidin, and its, similarity to the putative reaction intermediate, suggests it is a, transition state analog. This helps validate our contention that the role, of Asp169 is to electrostatically stabilize the reaction transition state.
+Allosamidin is a known inhibitor of class 18 chitinases. We show that allosamidin is a competitive inhibitor of the fungal chitinase CiX1 from Coccidioides immitis, with a K(i) of 60 nM. We report the X-ray structure of the complex and show that upon inhibitor binding the side-chain of Asp169 rotates to form an ion pair with the oxazolinium cation. The mechanism of action is thought to involve protonation of the leaving group by Glu171 and substrate assistance by the sugar acetamido moiety to form an oxazoline-like intermediate. We converted both amino acid residues to the corresponding amide and found that each mutation effectively abolishes enzyme activity. X-ray structures show the mutant enzymes retain the basic wild-type structure and that the loss of mutant activity is due to their altered chemical properties. The high affinity of allosamidin, and its similarity to the putative reaction intermediate, suggests it is a transition state analog. This helps validate our contention that the role of Asp169 is to electrostatically stabilize the reaction transition state.
 ==About this Structure==
-LL6 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Coccidioides_immitis Coccidioides immitis]. Active as [http://en.wikipedia.org/wiki/Chitinase Chitinase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.14 3.2.1.14] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1LL6 OCA].
+LL6 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Coccidioides_immitis Coccidioides immitis]. Active as [http://en.wikipedia.org/wiki/Chitinase Chitinase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.14 3.2.1.14] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1LL6 OCA].
 ==Reference==
@@ Line 16: / Line 16: @@
 [[Category: Bortone, K.]]
 [[Category: Ernst, S.]]
-[[Category: Monzingo, A.F.]]
+[[Category: Monzingo, A F.]]
-[[Category: Robertus, J.D.]]
+[[Category: Robertus, J D.]]
 [[Category: beta-alpha barrel]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 20:38:48 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:45:58 2008''