Homogenization and crystallization of histidine ammonia-lyase by exchange of a surface cysteine residue

Histidase (histidine ammonia-lyase, EC 4.3.1.3) from Pseudomonas putida was expressed in Escherichia coli and purified. In the absence of thiols the tetrameric enzyme gave rise to undefined aggregates and suitable crystals could not be obtained. The solvent accessibility along the chain was predicte...

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Bibliographic Details
Published in:Protein engineering 1999-02, Vol.12 (2), p.151-153
Main Authors: Schwede, Torsten F., Bädeker, Mathias, Langer, Martin, Rétey, Janos, Schulz, Georg E.
Format: Article
Language:English
Subjects:
Crystallography
Cysteine - chemistry
disulfide engineering
Ellman's reaction
Escherichia coli
histidine ammonia-lyase
Histidine Ammonia-Lyase - chemistry
Mutagenesis
Protein Structure, Secondary
Pseudomonas putida
Pseudomonas putida - enzymology
Time Factors
unspecific aggregation
X-ray crystallography
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Summary:Histidase (histidine ammonia-lyase, EC 4.3.1.3) from Pseudomonas putida was expressed in Escherichia coli and purified. In the absence of thiols the tetrameric enzyme gave rise to undefined aggregates and suitable crystals could not be obtained. The solvent accessibility along the chain was predicted from the amino acid sequence. Among the seven cysteines, only one was labeled as `solvent-exposed'. The exchange of this cysteine to alanine abolished all undefined aggregations and yielded readily crystals diffracting to 1.8 Å resolution.
ISSN:0269-2139
1741-0126
1460-213X
1741-0134
DOI:10.1093/protein/12.2.151