1fxh: Difference between revisions

Revision as of 13:43, 21 February 2008

MUTANT OF PENICILLIN ACYLASE IMPAIRED IN CATALYSIS WITH PHENYLACETIC ACID IN THE ACTIVE SITE

OverviewOverview

The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.

About this StructureAbout this Structure

1FXH is a Protein complex structure of sequences from Escherichia coli with and as ligands. Active as Penicillin amidase, with EC number 3.5.1.11 Full crystallographic information is available from OCA.

ReferenceReference

Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies., Alkema WB, Hensgens CM, Kroezinga EH, de Vries E, Floris R, van der Laan JM, Dijkstra BW, Janssen DB, Protein Eng. 2000 Dec;13(12):857-63. PMID:11239085

Page seeded by OCA on Thu Feb 21 12:43:45 2008

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-[[Image:1fxh.jpg|left|200px]]<br /><applet load="1fxh" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1fxh.jpg|left|200px]]<br /><applet load="1fxh" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1fxh, resolution 1.97&Aring;" />
 '''MUTANT OF PENICILLIN ACYLASE IMPAIRED IN CATALYSIS WITH PHENYLACETIC ACID IN THE ACTIVE SITE'''<br />
 ==Overview==
-The binding of penicillin to penicillin acylase was studied by X-ray, crystallography. The structure of the enzyme-substrate complex was, determined after soaking crystals of an inactive betaN241A penicillin, acylase mutant with penicillin G. Binding of the substrate induces a, conformational change, in which the side chains of alphaF146 and alphaR145, move away from the active site, which allows the enzyme to accommodate, penicillin G. In the resulting structure, the beta-lactam binding site is, formed by the side chains of alphaF146 and betaF71, which have van der, Waals interactions with the thiazolidine ring of penicillin G and the side, chain of alphaR145 that is connected to the carboxylate group of the, ligand by means of hydrogen bonding via two water molecules. The backbone, oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the, phenylacetic acid moiety through a bridging water molecule. Kinetic, studies revealed that the site-directed mutants alphaF146Y, alphaF146A and, alphaF146L all show significant changes in their interaction with the, beta-lactam substrates as compared with the wild type. The alphaF146Y, mutant had the same affinity for 6-aminopenicillanic acid as the wild-type, enzyme, but was not able to synthesize penicillin G from phenylacetamide, and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a, 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized, penicillin G more efficiently than the wild type. The combined results of, the structural and kinetic studies show the importance of alphaF146 in the, beta-lactam binding site and provide leads for engineering mutants with, improved synthetic properties.
+The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.
 ==About this Structure==
-FXH is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with CA and PAC as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Penicillin_amidase Penicillin amidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.1.11 3.5.1.11] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1FXH OCA].
+FXH is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=PAC:'>PAC</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Penicillin_amidase Penicillin amidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.1.11 3.5.1.11] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1FXH OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Penicillin amidase]]
 [[Category: Protein complex]]
-[[Category: Alkema, W.B.]]
+[[Category: Alkema, W B.]]
-[[Category: Dijkstra, B.W.]]
+[[Category: Dijkstra, B W.]]
 [[Category: Floris, R.]]
-[[Category: Hensgens, C.M.]]
+[[Category: Hensgens, C M.]]
-[[Category: Janssen, D.B.]]
+[[Category: Janssen, D B.]]
-[[Category: Kroezinga, E.H.]]
+[[Category: Kroezinga, E H.]]
-[[Category: Laan, J.M.van.der.]]
+[[Category: Laan, J M.van der.]]
-[[Category: Vries, E.de.]]
+[[Category: Vries, E de.]]
 [[Category: CA]]
 [[Category: PAC]]
 [[Category: ntn-hydrolase fold]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 15:26:08 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:43:45 2008''