1iw8
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(New page: 200px<br /><applet load="1iw8" size="450" color="white" frame="true" align="right" spinBox="true" caption="1iw8, resolution 2.5Å" /> '''Crystal Structure of ...)
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Revision as of 20:32, 24 November 2007
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Crystal Structure of a mutant of acid phosphatase from Escherichia blattae (G74D/I153T)
Overview
Escherichia blattae non-specific acid phosphatase (EB-NSAP) possesses a, pyrophosphate-nucleoside phosphotransferase activity, which is, C-5'-position selective. Current mutational and structural data were used, to generate a mutant EB-NSAP for a potential industrial application as an, effective and economical protein catalyst in synthesizing nucleotides from, nucleosides. First, Gly74 and Ile153 were replaced by Asp and Thr, respectively, since the corresponding replacements in the homologous, enzyme from Morganella morganii reduced the K(m) value for inosine and, thus increased the productivity of 5'-IMP. We determined the crystal, structure of G74D/I153T, which has a reduced K(m) value for inosine, as, expected. The tertiary structure of G74D/I153T was virtually identical to, that of the wild-type. In addition, neither of the introduced side chains, of Asp74 and Thr153 is directly involved in the interaction with inosine, in a hypothetical binding mode of inosine to EB-NSAP, although both, residues are situated near a potential inosine-binding site. These, findings suggested that a slight structural change caused by an amino acid, replacement around the potential inosine-binding site could significantly, reduce the K(m) value. Prompted by this hypothesis, we designed several, mutations and introduced them to G74D/I153T, to decrease the K(m) value, further. This strategy produced a S72F/G74D/I153T mutant with a 5.4-fold, lower K(m) value and a 2.7-fold higher V(max) value as compared to the, wild-type EB-NSAP.
About this Structure
1IW8 is a Single protein structure of sequence from Escherichia blattae with SO4 as ligand. Active as Acid phosphatase, with EC number 3.1.3.2 Full crystallographic information is available from OCA.
Reference
Enhancement of nucleoside phosphorylation activity in an acid phosphatase., Ishikawa K, Mihara Y, Shimba N, Ohtsu N, Kawasaki H, Suzuki E, Asano Y, Protein Eng. 2002 Jul;15(7):539-43. PMID:12200535
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