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PDBsum entry 1w5m
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References listed in PDB file
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Key reference
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Title
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Tracking the evolution of porphobilinogen synthase metal dependence in vitro.
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Authors
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F.Frère,
H.Reents,
W.D.Schubert,
D.W.Heinz,
D.Jahn.
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Ref.
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J Mol Biol, 2005,
345,
1059-1070.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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.
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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.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2005,
345,
1059-1070)
copyright 2005.
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