Biochemical Studies and Heterologous Expression of 1-Aminocyclopropane-1-Carboxylic Acid N-Malonyltransferase from Mung Hbean Hypocotyls

This dissertation, "Biochemical Studies and Heterologous Expression of 1-Aminocyclopropane-1-Carboxylic Acid N-Malonyltransferase From Mung Hbean Hypocotyls" by Cynthia, Leung Sau-wai, 梁秀慧, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled BIOCHEMICAL STUDIES AND HETEROLOGOUS EXPRESSION OF 1-AMINOCYCLOPROPANE-1- CARBOXYLIC ACID N-MALONYLTRANSFERASE FROM MUNG BEAN HYPOCOTYLS Submitted by LEUNG SAU WAI for the degree of Master of Philosophy at The University of Hong Kong in December 2002 1-Aminocyclopropane-1-carboxylic acid (ACC) N-malonyltransferase is one of the enzymes controlling the production of ethylene. It catalyzes the transfer of malonyl group from malonyl coenzyme A to ACC to form an involatile end product of malonyl ACC (MACC). The production of MACC provides an outlet for the accumulated ACC, reducing the generation of the plant hormone ethylene. ACC N-malonyltransferase was discovered in the 1980s and has been purified from different plants; however, its molecular properties have not yet been elucidated. Following the method suggested by Chick and Leung (1997), an immunopurified ACC N-malonyltransferase was obtained from mung bean hypocotyls. Its corresponding cDNA sequence (clone 11a) has been cloned previously (Man, 2000) and expression of the cDNA was investigated. Moreover, the deduced amino-acid sequence of clone 11a shows the presence of a catalytic triad of cysteine proteinase. Biochemical studies by two-dimensional gel electrophoresis, cyanogen bromide digestion, and N-terminal sequencing showed that the amino-acid sequence of the 40- kilodalton immunopurified ACC N-malonyltransferase agreed with that of clone 11a. The immunopurified enzyme also showed cysteine proteinase characteristics. In substrate-gel electrophoresis, the immunopurified ACC N-malonyltransferase could hydrolyze fibrinogen, gelatin, and lysozyme. Moreover, the proteolytic activity of the immunopurified ACC N-malonyltransferase on gelatin-substrate can be inhibited by 3- carboxy-trans-2, 3-epoxypropyl-leucylamido (4-guanidine) butane (E64), a cysteine proteinase inhibitor. On the other hand, the ACC N-malonyltransferase activity was not inhibited by E64. These data suggest that the immunopurified ACC N- malonyltransferase has two enzymatic activities. But whether they are contributed by one protein with two catalytic sites or from co-purified product has to be confirmed by heterologous expression. Despite the presence of a potential N-glycosylation site on the deduced amino-acid sequence of clone 11a, no glycosylation was detected. This suggests that ACC N-malonyltransferase would be a cystosolic enzyme. In heterologous expression studies of clone 11a, bacterial strains and yeast strains were employed. However, a low expression level was encountered in bacterial expression of the mature form of the enzyme in E. coli strains, BL21 (DE3), BL21 (DE3)   codon plus RIL and BL21 (DE3) codon plus RP. For yeast expression, three plasmids, pYES2_prepro, pYES2_pro and pYES2_mat, containing the sequence of the prepro- enzyme, the pro-enzyme and the mature enzyme, respectively, were constructed for expression in yeast strain Saccharomyces cerevisiae (INVSc1). Also, plasmid pESP1_mat, containing the mature form of clone 11a, was cloned for expression in yeast strain Schizosaccharomyces prombe (SPQ01). Purification was carried out on the recombinant protein of pESP1_mat under non-denaturing conditions. However, cysteine proteinase activity or ACC N-malonyltransferase activity remains undetected. Since neither