Details

Biochemical studies and heterologous expression of 1-Aminocyclopropane-1-Carboxylic Acid N-Malonyltransferase from mung hbean hypocotyls

by Leung Sau-wai, Cynthia

Abstract (Summary)
(Uncorrected OCR) Abstract of thesis entitled

BIOCHEMICAL STUDIES AND HETEROLOGOUS EXPRESSION OF lCARBOXYLIC ACID N-MALONYLTRANSFERASE FROM MUNG BEAN HYPOCOTYLS

Submitted by

LEUNGSAUWAI

for the degree of Master of Philosophy at The University of Hong Kong

in December 2002

l-Aminocyclopropane-l-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 lla) has been cloned previously (Man, 2000) and expression of the cDNA was investigated. Moreover, the deduced amino-acid sequence of clone lla 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 40kilo dalton immunopurified ACC N-malonyltransferase agreed with that of clone lla. 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 3carboxy-trans-2, 3-epoxypropyl-Ieucylamido (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 Nmalonyltransferase 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 lla, no glycosylation was detected. This suggests that ACC N-malonyltransferase would be a cystosolic enzyme.

In heterologous expression studies of clone lla, 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?IL and BL21 (DE3) codon plus?P. For yeast expression, three plasmids,

p YES2_prepro, p YES2_pro and p YES2_mat, containing the sequence of the preproenzyme, the pro-enzyme and the mature enzyme, respectively, were constructed for expression in yeast strain Saccharomyces cerevisiae (INVSc1). Also, plasmid pESPI_mat, containing the mature form of clone Ila, was cloned for expression in yeast strain Schizosaccharomyces prombe (SPQ01). Purification was carried out on the recombinant protein of pESPI_mat under non-denaturing conditions. However, cysteine proteinase activity or ACC N-malonyltransferase activity remains undetected.

Since neither ACC N-malonyltransferase nor cysteine proteinase activity were detected on heterologous expression. The ability of clone Iia needs to take further investigation. And the immunopurified ACC N-malonyltransferase may be a cysteine proteinase-like protein.

Bibliographical Information:

Advisor:

School:The University of Hong Kong

School Location:China - Hong Kong SAR

Source Type:Master's Thesis

Keywords:mung bean genetics enzymes gene expression

ISBN:

Date of Publication:01/01/2003

© 2009 OpenThesis.org. All Rights Reserved.