Skip Navigation

This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Omata, Y.
Right arrow Articles by Noguchi, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Omata, Y.
Right arrow Articles by Noguchi, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

J. Biochem, 2004, Vol. 136, No. 2 211-220
© 2004 The Japanese Biochemical Society

BIOCHEMISTRY

Purification and Characterization of Human Uroporphyrinogen III Synthase Expressed in Escherichia coli

Yoshiaki Omata, Hiroshi Sakamoto, Yuichiro Higashimoto, Shunsuke Hayashi and Masato Noguchi*

Department of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011

The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen (uro’gen) III synthase. Although the role of uro’gen III synthase is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of uro’gen III synthase have yet to be studied. We report here an expression system in Escherichia coli and a purification procedure for human uro’gen III synthase. The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of Cys residues that are accessible to 5,5'-dithiobis(2-nitrobenzoic acid) and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human uro’gen III synthase [Mathews et al. (2001) EMBO J. 20, 5832–5839], this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro’gen III synthase plays an important role in enzyme activity.

* To whom correspondence should be addressed. Tel: +81-942-31-7544, Fax: +81-942-31-4377, E-mail: mnoguchi{at}med.kurume-u.ac.jp


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.