1W5Q

Stepwise introduction of zinc binding site into porphobilinogen synthase of Pseudomonas aeruginosa (mutations A129C, D131C, D139C, P132E, K229R)

Summary for 1W5Q

• Entry DOI: 10.2210/pdb1w5q/pdb
• Related: 1B4K 1GZG 1W54 1W56 1W5M 1W5N 1W5O 1W5P
• Descriptor: DELTA-AMINOLEVULINIC ACID DEHYDRATASE, FORMIC ACID, MAGNESIUM ION, ... (6 entities in total)
• Functional Keywords: synthase, evolution, metalloenzyme, porphobilinogen synthase, pseudomonas aeruginosa, protein engineering
• Biological source: PSEUDOMONAS AERUGINOSA
• Total number of polymer chains: 2
• Total formula weight: 74654.54

Authors

Frere, F.,Reents, H.,Schubert, W.-D.,Heinz, D.W.,Jahn, D. (deposition date: 2004-08-09, release date: 2005-01-19, Last modification date: 2023-12-13)

Primary citation

Frere, F.,Reents, H.,Schubert, W.-D.,Heinz, D.W.,Jahn, D.
Tracking the Evolution of Porphobilinogen Synthase Metal Dependence in Vitro
J.Mol.Biol., 345:1059-, 2005

PubMed Abstract: 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.

PubMed: 15644204
DOI: 10.1016/J.JMB.2004.10.053

Experimental method

X-RAY DIFFRACTION (1.4 Å)