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PDBsum entry 2akc
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References listed in PDB file
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Key reference
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Title
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Structure and mutational analysis of the phon protein of salmonella typhimurium provide insight into mechanistic details.
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Authors
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R.D.Makde,
S.K.Mahajan,
V.Kumar.
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Ref.
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Biochemistry, 2007,
46,
2079-2090.
[DOI no: ]
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PubMed id
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Abstract
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The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and
belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We
report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate
complex, and the T159D mutant of PhoN along with functional characterization of
three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123,
Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and
tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The
T159D mutation, surprisingly, severely diminishes phosphatase activity,
apparently by disturbing the active site scaffold: Arg-191 is swung out of the
active site resulting in conformational changes in His-158 and His-197 residues.
Our results reveal a hitherto unknown functional role of Arg-191, namely,
restricting the active conformation of catalytic His-158 and His-197 residues.
Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the
D201N mutation completely abolished phosphatase activity. On the basis of this
observation and in silico analysis we suggest that the crucial mechanistic role
of Asp-201 is to stabilize the positive charge on the phosphohistidine
intermediate generated by the transfer of phosphoryl to the nucleophile,
His-197, located within hydrogen bond distance to the invariant Asp-201. This is
in contrast to earlier suggestions that Asp-201 stabilizes His-197 and the
His197-Asp201 dyad facilitates formation of the phosphoenzyme intermediate
through a charge-relay system. Finally, the L39T mutation in the conserved
polyproline motif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in
activity, in contrast to the nearly 50-fold reduction observed for monomeric
Prevotella intermedia acid phosphatase, suggesting that the varying quaternary
structure of PhoN orthologues may have functional significance.
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Secondary reference #1
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Title
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Purification, Crystallization and preliminary X-Ray diffraction studies of recombinant class a non-Specific acid phosphatase of salmonella typhimurium.
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Authors
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R.D.Makde,
V.Kumar,
A.S.Rao,
V.S.Yadava,
S.K.Mahajan.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 2003,
59,
515-518.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1 An alignment of chromosomal phoN of S. typhimurium
strain MD6001 (STM-KA) and the cloned gene (STM-CL). The
chromosomal phoN of S. typhimurium strain MD6001 is observed to
match exactly that of S. typhi (GenBank accession Nos. AF366353
and AL627282). The nucleotide bases and amino-acid residues
observed to differ in the chromosomal gene from the cloned
fragment are shown in bold lower case letters. The 20-residue
N-terminal signal sequence is underlined. The residues of the
conserved sequence motif,
KX[6]RP-(X[12-54])-PSGH-(X[31-54])-SRX[5]HX[3]D are identified
with asterisks.
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The above figure is
reproduced from the cited reference
with permission from the IUCr
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