This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, X.
Right arrow Articles by Xu, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.
Right arrow Articles by Xu, P.

 Previous Article  |  Next Article 

Journal of Bacteriology, December 2007, p. 9030-9036, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.01161-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Orotate Phosphoribosyltransferase from Corynebacterium ammoniagenes Lacking a Conserved Lysine{triangledown} ,{dagger}

Xing Wang,1,2 Cuiqing Ma,2 Xiuwen Wang,2 and Ping Xu1,2*

Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China,1 State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China2

Received 23 July 2007/ Accepted 26 September 2007

The pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase), was cloned by nested PCR and colony blotting from Corynebacterium ammoniagenes ATCC 6872, which is widely used in nucleotide production. Sequence analysis shows that there is a lack of an important conserved lysine (Lys 73 in Salmonella enterica serovar Typhimurium OPRTase) in the C. ammoniagenes OPRTase. This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinant Escherichia coli strain. The molecular mass of the purified OPRTase was determined to be 45.4 ± 1.5 kDa by gel filtration. Since the molecular mass for the subunit of the enzyme was 21.3 ± 0.6 kDa, the native enzyme exists as a dimer. Divalent magnesium was necessary for the activity of the enzyme and can be substituted for by Mn2+ and Co2+. The optimal pH for the forward (phosphoribosyl transfer) reaction is 10.5 to 11.5, which is higher than that of other reported OPRTases, and the optimal pH for the reverse (pyrophosphorolysis) reaction is 5.5 to 6.5. The Km values for the four substrates were determined to be 33 µM for orotate, 64 µM for 5-phosphoribosyl-1-pyrophosphate (PRPP), 45 µM for orotidine-5-phosphate (OMP), and 36 µM for pyrophosphate. The Km value for OMP is much larger than those of other organisms. These differences may be due to the absence of Lys 73, which is present in the active sites of other OPRTases and is known to interact with OMP and PRPP.

* Corresponding author. Mailing address: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China. Phone: 86(531)88564003. Fax: 86(531)88567250. E-mail: pingxu{at}sdu.edu.cn

{triangledown} Published ahead of print on 5 October 2007.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, December 2007, p. 9030-9036, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.01161-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»