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Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in Arabidopsis thaliana

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Abstract

Uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) are enzymes catalyzing the formation of uridine 5′-monophosphate (UMP) from uridine and adenine 5′-triphosphate (ATP) and from uracil and phosphoribosyl-α-1-pyrophosphate (PRPP), respectively, in the pyrimidine salvage pathway. Here, we report the characterization and functional analysis of a gene AtUK/UPRT1 from Arabidopsis thaliana. Sequencing of an expressed sequence tag clone of this gene revealed that it contains a full-length open reading frame of 1461 nucleotides and encodes a protein with a molecular mass of approximately 53 kDa. The sequence analysis revealed that the N-terminal region of AtUK/UPRT1 contains a UK domain and the C-terminal region consists of a UPRT domain. Expression of AtUK/UPRT1 in upp and upp-udk mutants of Escherichia coli supplied with 5-fluorouracil (5-FU) and 5-fluorouridine (5-FD) led to growth inhibition. Identical results were obtained with 5-FD and 5-FU treatments when the UK and UPRT domains were separated by the introduction of translation initiation and stop codons prior to complementation into the upp-udk and upp mutants. These results suggest that the AtUK/UPRT1 product can use uracil and uridine as substrates for the production of UMP. We also investigated the function of AtUK/UPRT1 in an Arabidopsis mutant. The wild-type Arabidopsis plants showed drastic growth retardation when they were treated with 5-FU and 5-FD while the growth of atuk/uprt1 mutant plants was not significantly affected. These findings confirm that AtUK/UPRT1 has a dual role in coding for both uridine kinase and uracil phosphoribosyltransferase that form UMP through the pyrimidine salvage pathway in Arabidopsis.

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Abbreviations

5-FU:

5-Fluorouracil

5-FD:

5-Fluorouridine

UK:

Uridine kinase

UPRT:

Uracil phosphoribosyltransferase

UMP:

Uridine 5′-monophosphate

ATP:

Adenine 5′-triphosphate

PRPP:

Phosphoribosyl-α-1-pyrophosphate

5-FUMP:

5-Fluoro-uridine monophosphate

5-FUDP:

5-Fluoro-uridine diphosphate

5-FUTP:

5-Fluoro-uridine triphosphate

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Acknowledgements

We wish to thank Prof. Dr. Donald J. Armstrong, Oregon State University, Corvallis, OR, USA for his valuable suggestions and critical reviewing of this manuscript. We like to thank Arabidopsis Biological Resource Center (ABRC) at the Ohio State University and E. coli Genetic Stock Center (CGSC) at Yale University for providing an EST clone (BE521919) and several E. coli upp, and upp-udk mutants, respectively. This work was supported by a Brain Korea 21 Research Fellowship from the Ministry of Education and Human Resources Development and BioGreen 21 from Rural Development Administration, Republic of Korea.

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  1. M. Rafiqul Islam

    Present address: Department of Soil Science, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh

  2. Shin-Wook Kang

    Present address: Laboratory of Equine Genetics, Korea Racing Association, Gyeonggi-do, 427-711, Republic of Korea

Authors and Affiliations

  1. Department of Biological Sciences, Seoul National University, Seoul, 151-742, Republic of Korea

    M. Rafiqul Islam, Hoyeun Kim, Shin-Wook Kang, Young-Min Jeong, Hyun-Ju Hwang, So-Young Lee & Sang-Gu Kim

  2. Department of Applied Biotechnology, Cheju National University, Jeju, 690-756, Republic of Korea

    Jung-Sup Kim

  3. Department of Biology, Mokpo National University, Jeonnam, 534-729, Republic of Korea

    Je-Chang Woo

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  1. M. Rafiqul Islam
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Correspondence to Sang-Gu Kim.

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Islam, M.R., Kim, H., Kang, SW. et al. Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in Arabidopsis thaliana . Plant Mol Biol 63, 465–477 (2007). https://doi.org/10.1007/s11103-006-9101-3

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