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| - | [[Image:1w56.gif|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_1w56| PDB=1w56 | SCENE= }} | | {{STRUCTURE_1w56| PDB=1w56 | SCENE= }} |
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| - | '''STEPWISE INTRODUCTION OF ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE OF PSEUDOMONAS AERUGINOSA (MUTATIONS A129C AND D131C)'''
| + | ===STEPWISE INTRODUCTION OF ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE OF PSEUDOMONAS AERUGINOSA (MUTATIONS A129C AND D131C)=== |
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| - | ==Overview==
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| - | 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.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_15644204}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 15644204 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_15644204}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Protein engineering]] | | [[Category: Protein engineering]] |
| | [[Category: Pseudomonas aeruginosa]] | | [[Category: Pseudomonas aeruginosa]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 13:10:35 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 09:20:51 2008'' |
Revision as of 06:20, 29 July 2008
Template:STRUCTURE 1w56
STEPWISE INTRODUCTION OF ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE OF PSEUDOMONAS AERUGINOSA (MUTATIONS A129C AND D131C)
Template:ABSTRACT PUBMED 15644204
About this Structure
1W56 is a Single protein structure of sequence from Pseudomonas aeruginosa. Full crystallographic information is available from OCA.
Reference
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
Page seeded by OCA on Tue Jul 29 09:20:51 2008
