PDBsum entry: 1iw8

Hydrolase

PDB id: 1iw8

Contents

Protein chains

(+ 0 more) 217 a.a. *

Ligands

SO4 ×6

Waters ×314

* Residue conservation analysis

PDB id:

1iw8

Name:

Hydrolase

Title:

Crystal structure of a mutant of acid phosphatase from escherichia blattae (g74d/i153t)

Structure:

Acid phosphatase. Chain: a, b, c, d, e, f. Fragment: residues 1-231. Engineered: yes. Mutation: yes

Source:

Escherichia blattae. Organism_taxid: 563. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Hexamer (from PQS)

Resolution:

2.50Å R-factor: 0.243 R-free: 0.284

Authors:

K.Ishikawa,Y.Mihara,N.Shimba,N.Ohtsu,H.Kawasaki,E.Suzuki, Y.Asano

Key ref:

K.Ishikawa et al. (2002). Enhancement of nucleoside phosphorylation activity in an acid phosphatase. Protein Eng, 15, 539-543. PubMed id: 12200535 DOI: 10.1093/protein/15.7.539

Date:

22-Apr-02 Release date: 11-Sep-02

Protein chains

Q9S1A6  (Q9S1A6_ESCBL) -  Acid phosphatase

Seq: 249 a.a.

Struc: 217 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.3.2 - Acid phosphatase.
• Reaction: A phosphate monoester + H₂O = an alcohol + phosphate

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

 Gene Ontology (GO) functional annotation 

• Cellular component: membrane (2 terms)
• Biochemical function: catalytic activity (3 terms)

reference

DOI no: 10.1093/protein/15.7.539

Protein Eng 15:539-543 (2002)

PubMed id: 12200535

Enhancement of nucleoside phosphorylation activity in an acid phosphatase.

K.Ishikawa, Y.Mihara, N.Shimba, N.Ohtsu, H.Kawasaki, E.Suzuki, Y.Asano.

ABSTRACT

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.