1w54: Difference between revisions
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[[Image:1w54.gif|left|200px]]<br /><applet load="1w54" size=" | [[Image:1w54.gif|left|200px]]<br /><applet load="1w54" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1w54, resolution 2.20Å" /> | caption="1w54, resolution 2.20Å" /> | ||
'''STEPWISE INTRODUCTION OF A ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE FROM PSEUDOMONAS AERUGINOSA (MUTATION D139C)'''<br /> | '''STEPWISE INTRODUCTION OF A ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE FROM PSEUDOMONAS AERUGINOSA (MUTATION D139C)'''<br /> | ||
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==About this Structure== | ==About this Structure== | ||
1W54 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_aeruginosa Pseudomonas aeruginosa] with K, MG, ZN and FMT as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Porphobilinogen_synthase Porphobilinogen synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.24 4.2.1.24] Known structural/functional Site: <scene name='pdbsite=AC1:Zn Binding Site For Chain B'>AC1</scene>. Full crystallographic information is available from [http:// | 1W54 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_aeruginosa Pseudomonas aeruginosa] with <scene name='pdbligand=K:'>K</scene>, <scene name='pdbligand=MG:'>MG</scene>, <scene name='pdbligand=ZN:'>ZN</scene> and <scene name='pdbligand=FMT:'>FMT</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Porphobilinogen_synthase Porphobilinogen synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.24 4.2.1.24] Known structural/functional Site: <scene name='pdbsite=AC1:Zn+Binding+Site+For+Chain+B'>AC1</scene>. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1W54 OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: pseudomonas aeruginosa]] | [[Category: pseudomonas aeruginosa]] | ||
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Revision as of 11:19, 3 February 2008
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STEPWISE INTRODUCTION OF A ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE FROM PSEUDOMONAS AERUGINOSA (MUTATION D139C)
OverviewOverview
Metal ions are indispensable cofactors for chemical catalysis by a, plethora of enzymes. Porphobilinogen synthases (PBGSs), which catalyse the, second step of tetrapyrrole biosynthesis, are grouped according to their, dependence on Zn(2+). Using site-directed mutagenesis, we embarked on, transforming Zn(2+)-independent Pseudomonas aeruginosa PBGS into a, Zn(2+)-dependent enzyme. Nine PBGS variants were generated by, permutationally introducing three cysteine residues and a further two, residues into the active site of the enzyme to match the homologous, Zn(2+)-containing PBGS from Escherichia coli. Crystal structures of seven, enzyme variants were solved to elucidate the nature of Zn(2+) coordination, at high resolution. The three single-cysteine variants were invariably, found to be enzymatically inactive and only one (D139C) was found to bind, detectable amounts of Zn(2+). The double mutant A129C/D139C is, enzymatically active and binds Zn(2+) in a tetrahedral coordination., Structurally and functionally it mimics mycobacterial PBGS, which bears an, equivalent Zn(2+)-coordination site. The remaining two double mutants, without known natural equivalents, reveal strongly distorted tetrahedral, Zn(2+)-binding sites. Variant A129C/D131C possesses weak PBGS activity, while D131C/D139C is inactive. The triple mutant A129C/D131C/D139C, finally, displays an almost ideal tetrahedral Zn(2+)-binding geometry and, a significant Zn(2+)-dependent enzymatic activity. Two additional amino, acid exchanges further optimize the active site architecture towards the, E.coli enzyme with an additional increase in activity. Our study, delineates the potential evolutionary path between Zn(2+)-free and, Zn(2+)-dependent PBGS enyzmes showing that the rigid backbone of PBGS, enzymes is an ideal framework to create or eliminate metal dependence, through a limited number of amino acid exchanges.
About this StructureAbout this Structure
1W54 is a Single protein structure of sequence from Pseudomonas aeruginosa with , , and as ligands. Active as Porphobilinogen synthase, with EC number 4.2.1.24 Known structural/functional Site: . Full crystallographic information is available from OCA.
ReferenceReference
Tracking the evolution of porphobilinogen synthase metal dependence in vitro., Frere F, Reents H, Schubert WD, Heinz DW, Jahn D, J Mol Biol. 2005 Feb 4;345(5):1059-70. Epub 2004 Dec 21. PMID:15644204
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