Molecular cloning and in silico analysis of a GTP cyclohydrolase I gene from grape ‎

Document Type : Original research paper


1 Department of Biotechnology Engineering, Faculty of Agricultural and Natural Resources, Imam ‎Khomeini International University, Qazvin, Iran‎

2 Department of Biotechnology Engineering, Faculty of Agricultural and Natural Resources, Imam ‎Khomeini International University, Qazvin, Iran‎


An entire open reading frame (ORF) encoding for a polypeptide of GTP cyclohydrolase I (GTPCH I) was isolated and cloned from Askari cultivar of grape (Vitis vinifera L.) berries. The 1,338-nucleotide ORF yields a 445-residue amino acid sequence with a calculated molecular mass of 48.65 kDa and a predicted isoelectric point of 6.43. The Vvgtpch I genomic sequence with a length of 4,964 bp contains two exons (169 and 1,169 bp) and an intron (2,676 bp). The gtpch I sequence of grape displayed a strong similarity with gtpch I sequence found in other plants, including peach (72%), cocoa (72%), strawberry (70%), and poplar (69%). Analysis of mRNA secondary structure revealed that the start codon of Vvgtpch I is completely exposed, suggesting a robust binding of the ribosome and efficient translation. Similar to gtpchs I from  diverse sources, molecular modeling uncovered that the monomer of VvGTPCH I adopts an αβ structure, which includes 10 α-helices and 8 β-sheets. Moreover, in silico analysis of the Vvgtpch I gene promoter identified potential cis-acting elements  responsive to environmental signals. This suggests that the Vvgtpch I gene has the capacity to be responsive to various environmental cues, such as heat, heavy metals, light, and plant hormones.


Agyenim-Boateng, K.G., Zhang, S., Shohag, M.J.I., Shaibu, A.S., Li, J., Li, B., and Sun, J. (2023). Folate biofortification in soybean: challenges and prospects. Agronomy 13(1): 241.
Amin, J., Ananthan, J., and Voellmy, R. (1988). Key features of heat shock regulatory elements. Mol Cell Biol 8(9): 3761-3769.
Auerbach, G., Herrmann, A., Bracher, A., Bader, G., Gütlich, M., Fischer, M., Neukamm, M., Garrido-Franco, M., Richardson, J., and Nar, H. (2000). Zinc plays a key role in human and bacterial GTP cyclohydrolase I. Proc Natl Acad Sci 97(25): 13567-13572.
Basset, G., Quinlivan, E.P., Ziemak, M.J., Díaz de la Garza, R., Fischer, M., Schiffmann, S., Bacher, A., Gregory III, J.F., and Hanson, A.D. (2002). Folate synthesis in plants: the first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I. Proc Natl Acad Sci 99(19): 12489-12494.
Basset, G.J., Quinlivan, E.P., Ravanel, S., Rébeillé, F., Nichols, B.P., Shinozaki, K., Seki, M., Adams-Phillips, L.C., Giovannoni, J.J., and Gregory III, J.F. (2004). Folate synthesis in plants: the p-aminobenzoate branch is initiated by a bifunctional PabA-PabB protein that is targeted to plastids. Proc Natl Acad Sci 101(6): 1496-1501.
Blancquaert, D., De Steur, H., Gellynck, X., and Van Der Straeten, D. (2014). Present and future of folate biofortification of crop plants. J Exp Bot 65(4): 895-906. doi: 10.1093/jxb/ert483.
Blancquaert, D., Van Daele, J., Strobbe, S., Kiekens, F., Storozhenko, S., De Steur, H., Gellynck, X., Lambert, W., Stove, C., and Van Der Straeten, D. (2015). Improving folate (vitamin B9) stability in biofortified rice through metabolic engineering. Nat Biotechnol 33(10): 1076-1078.
Blau, N., and van Spronsen, F.J. (2013). "Disorders of phenylalanine and tetrahydrobiopterin metabolism," in Physician's guide to the diagnosis, treatment, and follow-up of inherited metabolic diseases. Springer), 3-21.
Claverie, J.-M., and Notredame, C. (2011). Bioinformatics for dummies. John Wiley & Sons.
de Smit, M.H., and van Duin, J. (1990). Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. Proc Natl Acad Sci 87(19): 7668-7672.
Dong, W., Cheng, Z.-j., Lei, C.-l., Wang, X.-l., Wang, J.-l., Wang, J., Wu, F.-q., Zhang, X., Guo, X.-p., and Zhai, H.-q. (2014). Overexpression of folate biosynthesis genes in rice (Oryza sativa L.) and evaluation of their impact on seed folate content. Plant Foods Hum Nutr 69: 379-385.
Gallie, D.R. (1993). Posttranscriptional regulation of gene expression in plants. Annu Rev Plant Biol 44(1): 77-105.
Gofir, A., Wibowo, S., Hakimi, M., Putera, D.D., and Satriotomo, I. (2022). Folic acid treatment for patients with vascular cognitive impairment: a systematic review and meta-analysis. Int J Neuropsychopharmacol 25(2): 136-143.
Goldsbrough, A.P., Albrecht, H., and Stratford, R. (1993). Salicylic acid‐inducible binding of a tobacco nuclear protein to a 10 bp sequence which is highly conserved amongst stress‐inducible genes. Plant J 3(4): 563-571.
Gorelova, V., Ambach, L., Rébeillé, F., Stove, C., and Van Der Straeten, D. (2017). Folates in plants: research advances and progress in crop biofortification. Front Chem 5: 21.
Gräwert, T., Fischer, M., and Bacher, A. (2013). Structures and reaction mechanisms of GTP cyclohydrolases. IUBMB life 65(4): 310-322.
Japelaghi, R.H., Haddad, R., and Garoosi, G.-A. (2011). Rapid and efficient isolation of high quality nucleic acids from plant tissues rich in polyphenols and polysaccharides. Mol Biotechnol 49: 129-137.
Liang, Q., Wang, K., Liu, X., Riaz, B., Jiang, L., Wan, X., Ye, X., and Zhang, C. (2019). Improved folate accumulation in genetically modified maize and wheat. J Exp Bot 70(5): 1539-1551.
Maier, J., Witter, K., Gutlich, M., Ziegler, I., Werner, T., and Ninnemann, H. (1995). Homology cloning of GTP-cyclohydrolase-I from various unrelated eukaryotes by reverse-transcription polymerase chain-reaction using a general set of degenerate primers. Biochem Biophys Res Commun 212(2): 705-711.
Maita, N., Hatakeyama, K., Okada, K., and Hakoshima, T. (2004). Structural basis of biopterin-induced inhibition of GTP cyclohydrolase I by GFRP, its feedback regulatory protein. J Biol Chem 279(49): 51534-51540.
Meyer, Y., Vignols, F., and Reichheld, J.P. (2002). Classification of plant thioredoxins by sequence similarity and intron position. Meth Enzymol 347: 394-402.
Morowvat, M.H., Babaeipour, V., Rajabi-Memari, H., Vahidi, H., and Maghsoudi, N. (2014). Overexpression of recombinant human beta interferon (rhINF-β) in periplasmic space of Escherichia coli. Iran J Pharm Res 13(Suppl): 151.
Nar, H., Huber, R., Auerbach, G., Fischer, M., Hösl, C., Ritz, H., Bracher, A., Meining, W., Eberhardt, S., and Bacher, A. (1995). Active site topology and reaction mechanism of GTP cyclohydrolase I. Proc Natl Acad Sci 92(26): 12120-12125.
Nunes, A.C., Kalkmann, D.C., and Aragao, F.J. (2009). Folate biofortification of lettuce by expression of a codon optimized chicken GTP cyclohydrolase I gene. Transgenic Res 18(5): 661-667.
Prabhu, A.A., Veeranki, V.D., and Dsilva, S.J. (2016). Improving the production of human interferon gamma (hIFN-γ) in Pichia pastoris cell factory: An approach of cell level. Process Biochem 51(6): 709-718.
Proctor, J.R., and Meyer, I.M. (2013). C o F old: an RNA secondary structure prediction method that takes co-transcriptional folding into account. Nucleic Acids Res 41(9): e102-e102.
Ramírez Rivera, N.G., García‐Salinas, C., Aragão, F.J., and Díaz de la Garza, R.I. (2016). Metabolic engineering of folate and its precursors in Mexican common bean (Phaseolus vulgaris L.). Plant Biotechnol J 14(10): 2021-2032.
Rebelo, J., Auerbach, G., Bader, G., Bracher, A., Nar, H., Hösl, C., Schramek, N., Kaiser, J., Bacher, A., and Huber, R. (2003). Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I. J Mol Biol 326(2): 503-516.
Rossignol, D.A., and Frye, R.E. (2021). Cerebral folate deficiency, folate receptor alpha autoantibodies and leucovorin (folinic acid) treatment in autism spectrum disorders: a systematic review and meta-analysis. J Pers Med 11(11): 1141.
Sahr, T., Ravanel, S., and Rébeillé, F. (2005). Tetrahydrofolate biosynthesis and distribution in higher plants. Biochem 33(4): 758-762.
Sahrawy, M., Hecht, V., Lopez-Jaramillo, J., Chueca, A., Chartier, Y., and Meyer, Y. (1996). Intron position as an evolutionary marker of thioredoxins and thioredoxin domains. J Mol Evol 42: 422-431.
Shlobin, N.A., LoPresti, M.A., Du, R.Y., and Lam, S. (2020). Folate fortification and supplementation in prevention of folate-sensitive neural tube defects: a systematic review of policy. J Neurosurg Pediatr 27(3): 294-310.
Sigrist, C.J., Cerutti, L., Hulo, N., Gattiker, A., Falquet, L., Pagni, M., Bairoch, A., and Bucher, P. (2002). PROSITE: a documented database using patterns and profiles as motif descriptors. Brief Bioinformatics 3(3): 265-274.
Steinmetz, M.O., Plüss, C., Christen, U., Wolpensinger, B., Lustig, A., Werner, E.R., Wachter, H., Engel, A., Aebi, U., and Pfeilschifter, J. (1998). Rat GTP cyclohydrolase I is a homodecameric protein complex containing high-affinity calcium-binding sites. J Mol Biol 279(1): 189-199.
Strobbe, S., and Van Der Straeten, D. (2017). Folate biofortification in food crops. Curr Opin Biotechnol 44: 202-211.
Stuart, G.W., Searle, P.F., and Palmiter, R.D. (1985). Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences. Nature 317(6040): 828-831.
Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24(8): 1596-1599.
Volume 11, Issue 1
January 2023
Pages 1-16
  • Receive Date: 25 February 2023
  • Revise Date: 07 October 2023
  • Accept Date: 08 October 2023
  • First Publish Date: 08 October 2023