Sains Malaysiana 40(4)(2011): 331–337

Cloning and Analysis of pyrG Gene Encoding Orotidine 5-Monophosphate Decarboxylase of Aspergillus oryzae Strain S1

(Pengklonan dan Analisis Gen pyrG yang Mengekodkan Orotidina 5-monofosfat Dekarboksilase Aspergillus oryzae)

Selina Oh Siew Ling¹, Leong Jiun Min¹, Abdul Munir Abdul Murad¹, Nor Muhammad Mahadi² & Farah Diba Abu Bakar¹ *

¹School of BioSciences and Biotechnology, Faculty of Science and Technology

43600 Bangi, Selangor D.E., Malaysia

²Malaysia Genome Insitute, Helix Emas Block

UKM-MTDC Smart Technology Centre, Universiti Kebangsaan Malaysia

43600 Bangi, Selangor D.E., Malaysia

Diserahkan: 9 Disember 2009 / Diterima: 15 Julai 2010

ABSTRACT

In this study, the pyrG gene which encodes for orotidine 5-monophosphate decarboxylase (OMP decarboxylase) of Aspergillus oryzae strain S1 was cloned and analysed. This 1.8kb A. oryzae pyrG encompasses the 5’-regulatory flanking region (465 bp), open reading frame (899 bp) and 3’-regulatory region (475 bp). The pyrG contained one intron at position 623-687 bp based on the AUGUSTUS and FGENESH (SoftBerry) analysis corresponding to the intron present in the pyrG of A. oryzae (Accession Number: Y13811). In silico analysis showed that the enzyme encoded by the A. oryzae S1 pyrG gene has a theoretical molecular weight of 30.28 kDa and theoretical pI value of 5.92. This enzyme is hydrophilic, located in a region outside of the transmembrane and it functions in the cytoplasm. Five motives such as N-glycosylation site, protein kinase C (PKC) phosphorylation site, casein kinase II (CK-2) phosphorylation site, N-myristolation site and orotidine 5-monophoshate decarboxylase active site have been identified in the pyrG amino acid sequence. The three dimensional structure of this enzyme generated via protein homology modeling using the bioinformatic software, Swiss Model, shows that OMP decarboxylase is a protein with an α/ß barrel structure possessing 8 ß-strands surrounded by 9 α-helices. The amino acid residues involved in the active site have been identified and it is located on one of the ß-strands. The pyrG DNA sequence will be used for the complementation of a pyrG auxotroph mutant of A. oryzae.

Keywords: Aspergillus oryzae; orotidine 5-monophosphate dehydrogenase; pyrG

ABSTRAK

Dalam kajian ini, gen pyrG yang mengekod orotidina 5-monofosfat dekarboksilase (OMP dekarboksilase) Aspergillus oryzae strain S1 telah diklon dan dianalisis. Gen pyrG ~1.8 kb A. oryzae ini merangkumi kawasan pengawalaturan 5’ (465 pb), rangka bacaan terbuka (899 pb) dan kawasan pengawalaturan 3’ (475 pb). Gen pyrG ini mempunyai satu intron pada kedudukan 623-687 pb berdasarkan kepada analisis AUGUSTUS dan FGENESH (SoftBerry) bersamaan dengan kedudukan intron yang hadir dalam gen pyrG A. oryzae (Nombor Aksesi: Y13811). Analisis in silico menunjukkan bahawa enzim yang dikodkan oleh pyrG A. oryzae strain S1 mempunyai berat molekul teori sebanyak 30.28 kDa dan nilai pI teori bernilai 5.92. Enzim ini bersifat hidrofilik, berada di kawasan luar transmembran dan ia berfungsi di dalam sitoplasma sel. Lima motif telah dikenalpasti dalam jujukan asid amino pyrG iaitu tapak N-glikosilasi, tapak pemfosfatan protein kinase C (PKC), tapak pemfosfatan kasein kinase II (CK-2), tapak N-Miristolasi dan tapak aktif orotidina 5-monofosfat dekarboksilase. Struktur tiga dimensi enzim ini yang dijanakan menggunakan pendekatan pemodelan homologi protein melalui perisian bioinformatik Swiss Model menunjukkan bahawa OMP dekarboksilase adalah protein yang mempunyai struktur α/ß barrel dengan 8 kepingan struktur ß yang dikelilingi oleh 9 struktur heliks. Residu asid amino yang terlibat dalam tapak aktif telah dikenalpasti dan ia berada pada salah satu daripada kepingan struktur ß protein tersebut. Jujukan DNA pyrG ini akan digunakan untuk mengkomplementasikan mutan auksotrof pyrG A. oryzae.

Kata kunci: Aspergillus oryzae; orotidina 5-monofosfat dehidrogenase; pyrG

RUJUKAN

Arnold, K., Bordoli, L., Kopp, J. & Schwede, T. 2006. The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201.

Bairoch, A., Bucher, P. & Hofmann, K. 1997. The PROSITE database. Nucleic Acids Res. 25(1): 217-221.

Boeke, J.D., Lacroute, F. & Fink, G.R. 1984. A positive selection for mutants lacking orotidine-5’-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197: 345-346.

Gellison, G. 2005. Production of recombinant protein. Novel Microbial and Eucaryotic Expression System. Wiley-VCHVerlag GmbH & Co. KGaA: Weinheim

Hiller, K., Schobert, M., Hundertmark, C., Jahn, D. & Münch, R. 2003. JVirGel: calculation of virtual two dimensional protein gels. Nucleic Acids Res. 31: 3862-3865.

Jacquet, M., Guilbaud, R. & Garreau, H. 1988. Sequence analysis of the DdPYR5-6 gene coding for UMP synthase in Dictyostelium discoideum and comparison with orotate phosphoribosyl transferases and OMP decarboxylases. Mol. Gen. Genet. 211: 441-445.

Kobayashi, T., Abe, K., Asai, K., Gomi, K., Juvvadi, P.R., Kato, M., Kitamoto, K., Takeuchi, M. & Machida, M. 2007. Genomics of Aspergillus oryzae. Biosci. Biotechnol. Biochem. 71: 646-670.

Kyte, J. & Doolittle, R.F. 1982. Amino acid scale: hydropathicity. J. Mol. Biol. 157: 105-132.

Long, H., Wang, T.H. & Zhang, Y.K. 2008. Isolation of Trichoderma reesei pyrG Negative Mutant by UV Mutagenesis and Its Application in Transformation. Chem. Res. Chin. Univ. 24(5): 565-569.

Machida, M., Asai, K., Sano, M., Tanaka, T., Kumagai, T., Terai, G., Kusumoto, K., Arima, T., Akita, O., Kashiwagi, Y., Abe, K., Gomi, K., Horiuchi, H., Kitamoto, K., Kobayashi, T., Takeuchi, M., Denning, D.W., Galagan, J.E., Nierman, W.C., Yu, J., Archer, D.B., Bennett, J.W., Bhatnagar, D., Cleveland, T.E., Fedorova, N.D., Gotoh, O., Horikawa, H., Hosoyama, A., Ichinomiya, M., Igarashi, R., Iwashita, K., Juvvadi, P.R., Kato, M., Kato, Y., Kin, T., Kokubun, A., Maeda, H., Maeyama, N., Maruyama, J., Nagasaki, H., Nakajima, T., Oda, K., Okada, K., Paulsen, I., Sakamoto, K., Sawano, T., Takahashi, M., Takase, K., Terabayashi, Y., Wortman, J.R., Yamada, O., Yamagata, Y., Anazawa, H., Hata, Y., Koide, Y., Komori, T., Koyama, Y., Minetoki, T., Suharnan, S., Tanaka, A., Isono, K., Kuhara, S., Ogasawara, N. & Kikuchi, H. 2005. Genome sequencing and analysis of Aspergillus oryzae. Nature 438: 1157-1161.

Mattern, I.E., Unkles, S.E., Kinghorn, J.R., Pouwels, P.H. & van den Hondel C.A.M.J.J. 1987. Transformation of Aspergillus oryzae using the Aspergillus niger pyrG gene. Mol. Gen. Genet. 210: 460-461.

Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10: 1-6.

Pich, U. & Schubert, I. 1993. Mediprep method for isolation of DNA from plant samples with a high content of polyphenolics. Nucleic Acids Res. 21: 3328.

Stanke, M., Steinkamp, R., Waack, S. & Morgenstern, B. 2004. “AUGUSTUS: a web server for gene finding in eukaryotes” Nucleic Acids Res. 32: 309-312.

Ward, O.P., Qin, W.M., Dhanjoon, J., Ye, J. & Singh, A. 2006. Physiology and Biotechnology of Aspergillus. Advances in Applied Microbiology 58: 1-75.

Yolanda, M.J.T., de Ruiter-Jacobs, Martien, B. & Unkles, S.E. 1989. A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae. Current Genetics 16: 159-163.

*Pengarang untuk surat-menyurat; email: fabyff@ukm.my