AJPS  Vol.5 No.18 , August 2014
Cloning and Enzymatic Activity Analysis of the Malonyl-CoA:acyl Carrier Protein Transacylase in Brassica napus Cultivars with Different Oil Content
Abstract: The full-length genomic DNA of MCAT (Malonyl-CoA:acyl carrier protein transacylase) in Brassica napus was cloned. BnMCAT shares very high identity with AtMCAT in gene sequence and gene structure. A multiple alignment of the protein sequence showed that BnMCAT shares high identity with other MCATs from E. coli and plants. BnMCAT was expressed in all tissues, such as roots, stems, leaves, flowers, and seeds, and no significant differences in the expression level were found in different embryo stages after pollination. According to an in vitro relative activity analysis, purified recombinant BnMCAT expressed in E. coli had transacylase activity. Although the relative activities of BnMCAT in crude extracts isolated from different staged embryos were similar and showed little variation, a higher relative activity was found in a crude extract isolated from embryos in comparison to leaves. Different relative activities of BnMCAT in crude extracts isolated from cultivars with different oil content were also found, suggesting that the activity of BnMCAT might be a decisive factor for a high oil content. Together, these results showed that BnMCAT is an important enzyme in the FAS system and indicate that BnMCAT might be a new target enzyme for future crop improvement through genetic engineering.
Cite this paper: Qu, C. , Jiang, H. and Zhang, L. (2014) Cloning and Enzymatic Activity Analysis of the Malonyl-CoA:acyl Carrier Protein Transacylase in Brassica napus Cultivars with Different Oil Content. American Journal of Plant Sciences, 5, 2599-2610. doi: 10.4236/ajps.2014.518274.

[1]   White, S.W., Zheng, J., Zhang, Y.M. and Rock, C.O. (2005) The Structural Biology of Type II Fatty Acid Biosynthesis. Annual Review of Biochemistry, 74, 791-831.

[2]   Arthur, C.J., Szafranska, A., Evans, S.E., Findlow, S.C., Burston, S.G., Owen, P., Clark-Lewis, I., Simpson, T.J., Crosby, J. and Crump, M. (2005) Self-Malonylation Is an Intrinsic Property of a Chemically Synthesized Type II Polyketide Synthase Acyl Carrier Protein. Biochemisty, 44, 15414-15421.

[3]   Keatinge-Clay, A.T., Shelat, A.A., Savage, D.F., Tsai, S.C., Miercke, L.J., O’Connell, J.D., Khosla, C. and Stroud, R.M. (2003) Catalysis, Specificity, and ACP Docking Site of Streptomyces Coelicolor Malonyl-CoA: ACP Transacylase. Structure, 11, 147-154.

[4]   Summers, R.G., Ali, A., Shen, B., Wessel, W.A. and Hutchinson, C.R. (1995) Malonyl-Coenzyme A: Acyl Carrier Protein Acyltransferase of Streptomyces glaucescens: A Possible Link between Fatty Acid and Polyketide Biosynthesis. Biochemistry, 34, 9389-9402.

[5]   Simon, J.W. and Slabas, A.R. (1998) cDNA Cloning of Brassica Napus Malonyl-CoA:ACP Transacylase (MCAT) (fab D) and Complementation of an E. coli MCAT Mutant. FEBS Letters, 435, 204-206.

[6]   Hajduch, M., Casteel, J.E., Hurrelmeyer, K.E., Song, A., Agrawal, G.K. and Thelen, J.J. (2006) Proteomic Analysis of Seed Filling in Brassica napus. Developmental Characterization of Metabolic Isozymes Using High-Resolution Two-Dimensional Gel Electrophoresis. Plant Physiology, 141, 32-46.

[7]   Martínez-Force, E.,álvarez-Ortega, R. and Garcés, R. (1999) Enzymatic Characterisation of High-Palmitic Acid Sunflower (Helianthus annuus L.) Mutants. Planta, 207, 533-538.

[8]   Kremer, L., Nampoothiri, K.M., Lesjean, S., Dover, L.G., Graham, S., Betts, J., Brennan, P.J., Minnikin, D.E., Locht, C. and Besra, G.S. (2001) Biochemical Characterization of Acyl Carrier Protein (AcpM) and Malonyl-CoA: AcpM Transacylase (mtFabD), Two Major Components of Mycobacterium tuberculosis Fatty Acid Synthase II. The Journal of Biological Chemistry, 276, 27967-27974.

[9]   Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantitative of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72, 248-254.

[10]   Serre, L., Verbree, E.C., Dauter, Z., Stuije, A.R. and Derewenda, Z.S. (1995) The Escherichia coli Malonyl-CoA: Acyl Carrier Protein Transacylase at 1.5-A Resolution. Crystal Structure of a Fatty Acid Synthase Component. The Journal of Biological Chemistry, 270, 12961-12964.

[11]   Harwood, J.L. (1996) Recent Advances in the Biosynthesis of Plant Fatty Acids. Biochimica et Bioohysica Acta, 130, 7-56.

[12]   Verwoert, I.I., van der Linden, K.H., Nijkamp, H.J. and Stuitje, A.R. (1994) Developmental Specific Expression and Organelle Targeting of the Escherichia coli fabD Gene, Encoding Malonyl Coenzyme A-Acyl Carrier Protein Transacylase in Transgenic Rape and Tobacco Seeds. Plant Molecular Biology, 26, 189-202.

[13]   Stapleton, S.R. and Jaworski, J.G. (1984) Characterization and Purfication of Malonyl-Coenzyme A: [Acyl-carrierprotein] Transacylases from Spinach and Anabaena variabilis. Biochimica et Biophysica Acta, 794, 240-248.

[14]   Guerra, D.J. and Ohlrogge, J.B. (1986) Partial Purification and Characterization of Two Forms of Malonyl-Coenzyme A: Acyl Carrier Proteintransacylase from Soybean Leaf Tissue. Archives of Biochemistry and Biophysics, 246, 274-285.

[15]   He, Y.-Q. and Wu, Y. (2009) Oil Body Biogenesis during Brassica napus Embryogenesis. Journal of Integrative Plant Biology, 51, 792-799.

[16]   Nelson, D.L. and Cox, M.M. (2008) Principles of Biochemistry. 5th Edition, W. H. Freeman and Company, New York.

[17]   Oefner, C., Schulz, H., D’Arcy, A. and Dale, G.E. (2006) Mapping the Active Site of Escherichia coli Malonyl-CoA-acyl Carrier Protein Transacylase (FabD) by Protein Crystallography. Acta Crystallographica Section D: Biological Crystallography, 62, 613-618.

[18]   Zhang, L., Liu, W., Xiao, J., Hu, T., Chen, J., Chen, K., Jiang, H. and Shen, X. (2007) Malonyl-CoA:Acyl Carrier Protein Transacylase from Helicobacter pylori: Crystal Structure and Its Interaction with Acyl Carrier Protein. Protein Science, 16, 1184-1192.

[19]   Li, Z., Huang, Y., Ge, J., Fan, H., Zhou, X., Li, S., Bartlam, M., Wang, H. and Rao, Z. (2007) The Crystal Structure of MCAT from Mycobacterium tuberculosis Reveals Three New Catalytic Models. Journal of Molecular Biology, 371, 1075-1083.

[20]   Hong, S.K., Kim, K.H. and Kim, E.E. (2010) Cloning, Purification, Crystallization and Preliminary X-Ray Crystallographic Analysis of MCAT from Staphylococcus Aureus. Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 66, 20-22.

[21]   Kim, T.O., Im, D.W., Jung, H.J., Kwon, S.J. and Heo, Y.S. (2012) Purification, Crystallization and Preliminary X-Ray Diffraction Analysis of Enoyl-Acyl Carrier Protein Reductase (FabK) from Streptococcus mutans Strain UA159. Acta Crystallographica, 68, 292-294.

[22]   Natarajan, S., Kim, J.K., Jung, T.K., Doan, T.T., Ngo, H.P., Hong, M.K., Kim, S., Tan, V.P., Ahn, S.J., Lee, S.H., Han, Y., Ahn, Y.J. and Kang, L.W. (2012) Crystal Structure Of Malonyl CoA-Acyl Carrier Protein Transacylase from Xanthomanous oryzae pv. oryzae and Its Proposed Binding with ACP. Molecules and Cells, 33, 19-25.

[23]   Baldock, C., Rafferty, J.B., Sedelnikova, S.E., Baker, P.J., Stuitje, A.R., Slabas, A.R., Hawkes, T.R. and Rice, D.W. (1996) A Mechanism of Drug Action Revealed by Structural Studies of Enoyl Reductase. Science, 274, 2108-2110.

[24]   Shimakata, T. and Stumpf, P.K. (1983) The Purification and Function of Acetyl Coenzyme A: Acyl Carrier Protein Transacylase. The Journal of Biological Chemistry, 258, 3592-3598.

[25]   Arthur, C.J., Szafranska, A.E., Long, J., Mills, J., Cox, R.J., Findlow, S.C., Simpson, T.J., Crump, M.P. and Crosby, J. (2006) The Malonyl Transferase Activity of Type II Polyketide Synthase Acyl Carrier Proteins. Chemistry & Biology, 13, 587-596.

[26]   Misra, A., Sharma, S.K., Surolia, N. and Surolia, A. (2007) Self-Acylation Properties of Type II Fatty Acid Biosynthesis Acyl Carrier Protein. Chemistry & Biology, 14, 775-783.

[27]   Misra, A., Surolia, N. and Surolia, A. (2009) Catalysis and Mechanism of Malonyl Transferase Activity in Type II Fatty Acid Biosynthesis Acyl Carrier Proteins. Molecular BioSystems, 5, 651-659.

[28]   Florova, G., Kazanina, G. and Reynolds, K.A. (2002) Enzymes Involved in Fatty Acid and Polyketide Biosynthesis in Streptomyces glaucescens: Role of FabH and FabD and Their Acyl Carrier Protein Specificity. Biochemistry, 41, 10462-10471.

[29]   Molnos, J., Gardiner, R., Dale, G.E. and Lange, R. (2003) A Continuous Coupled Enzyme Assay for Bacterial Malonyl-CoA: Acyl Carrier Protein Transacylase (FabD). Analytical Biochemistry, 319, 171-176.

[30]   Jeon, E., Lee, S., Won, J.I., Han, S.O., Kim, J. and Lee, J. (2011) Development of Escherichia coli MG1655 Strains to Produce Long Chain Fatty Acids by Engineering Fatty Acid Synthesis (FAS) Metabolism. Enzyme and Microbial Technology, 49, 44-51.

[31]   Zhang, X., Agrawal, A. and San, K.Y. (2012) Improving Fatty Acid Production in Escherichia coli through the Overexpression of Malony CoA-Acyl Carrier Protein Transacylase. Biotechnology Progress, 28, 60-65.