PDBsum entry 1w5m

Synthase

PDB id: 1w5m

Contents

Protein chains

330 a.a. *

Ligands

FMT ×2

Metals

_MG ×7

_CL ×2

_ZN ×2

Waters ×887

* Residue conservation analysis

PDB id:

1w5m

Name:

Synthase

Title:

Stepwise introduction of zinc binding site into porphobilinogen synthase of pseudomonas aeruginosa (mutations a129c and d139c)

Structure:

Delta-aminolevulinic acid dehydratase. Chain: a, b. Synonym: porphobilinogen synthase, alad, aladhporphobilinogen synthase. Engineered: yes. Mutation: yes

Source:

Pseudomonas aeruginosa. Organism_taxid: 208964. Strain: pao1. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Biol. unit:

Octamer (from PDB file)

Resolution:

1.60Å R-factor: 0.131 R-free: 0.165

Authors:

F.Frere,H.Reents,W.-D.Schubert,D.W.Heinz,D.Jahn

Key ref:

F.Frère et al. (2005). Tracking the evolution of porphobilinogen synthase metal dependence in vitro. J Mol Biol, 345, 1059-1070. PubMed id: 15644204 DOI: 10.1016/j.jmb.2004.10.053

Date:

09-Aug-04 Release date: 19-Jan-05

Protein chains

Q59643  (HEM2_PSEAE) -  Delta-aminolevulinic acid dehydratase from Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)

Seq: 337 a.a.

Struc: 330 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.4.2.1.24 - porphobilinogen synthase.
• Pathway: Porphyrin Biosynthesis (early stages)
• Reaction: 2 5-aminolevulinate = porphobilinogen + 2 H2O + H⁺
• Cofactor: Zn(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1016/j.jmb.2004.10.053

J Mol Biol 345:1059-1070 (2005)

PubMed id: 15644204

Tracking the evolution of porphobilinogen synthase metal dependence in vitro.

F.Frère, H.Reents, W.D.Schubert, D.W.Heinz, D.Jahn.

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.

Selected figure(s)

Figure 1.
Figure 1. Enzymatic reaction catalyzed by PBGS during tetrapyrrole biosynthesis. Two molecules of ALA are condensed asymmetrically to form the pyrrole derivative PBG.

Figure 3.
Figure 3. Schematic representation of the active sites of crystallized mutant enzymes delineating the transition of PaPBGS to the Zn2+-binding, EcPBGS-like mutant CCC. Residue, substrate and background color coding is identical with that in Figure 2.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 345, 1059-1070) copyright 2005.

Figures were selected by an automated process.