Skip Navigation



Journal of Biochemistry Advance Access published online on October 20, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp162
This Article
Right arrow Advance Access manuscript (PDF)
Right arrow Supplementary Data
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 Tsutsumi, M.
Right arrow Articles by Kano, K.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsutsumi, M.
Right arrow Articles by Kano, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Site-directed Mutation at Residues near the Catalytic Site of Histamine Dehydrogenase from Nocardioides simplex and Its Effects on Substrate Inhibition

Maiko Tsutsumi, Noriaki Tsuse, Nobutaka Fujieda** and Kenji Kano*

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

*To whom correspondence should be addressed: Dr. Kano, Kenji Tel: +81-75-753-6392, Fax: +81-75-753-6456, E-mail: kkano{at}kais.kyoto-u.ac.jp

Received September 16, 2009; Accepted October 4, 2009


  Abstract

Histamine dehydrogenase from Nocardioides simplex (nHmDH) is a homodimer containing one 6-S-cysteinyl FMN (CFMN) and one [4Fe-4S] cluster per monomer. nHmDH catalyzes the oxidative deamination of histamine to ammonia and imidazole acetaldehyde, but histamine inhibits its catalytic activity at high concentrations. We mutated gene encoded residues (Tyr180, Gly268 and Asp269) near CFMN to understand the biophysical meaning of the substrate inhibition. Three mutants Y180F, G268D/D269C and Y180F/G268D/D269C were expressed by considering the DNA sequence alignment of histamine dehydrogenase from Rhizobium sp. 4-9 (rHmDH), which does not suffer from the substrate inhibition. The Y180F/G268D/D269C mutation to mimic rHmDH successfully suppressed the inhibition, although the catalytic activity decreased. The substrate inhibition was weakened by the Y180F mutation, but G268D/D269C was still susceptible to the inhibition. It was found that also causes changes in the UV-vis absorption spectra of the substrate-reduced form and the redox potential of the enzymes. The characterization suggests that the thermodynamic preference of the semiquinone form of CFMN in the two-electron-reduced subunit of the enzyme is responsible for the substrate inhibition. However, destabilization of the semiquinone form leads to kinetic hindrance due to the uphill single electron transfer from the fully reduced CFMN to the [4Fe-4S] cluster.

Key Words: histamine dehydrogenase, substrate inhibition, 6-S-cysteinyl flavin mononucleotide, [4Fe-4S] iron-sulfur cluster, redox potential

** Present address: Department of Material and Life Science, Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan


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.