PolyU IR
 

PolyU Institutional Repository >
Applied Biology and Chemical Technology >
ABCT Theses >

Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/3301

Title: Properties of novel pyridoxal kinase enzymes
Authors: Kwok, Sui-yi
Subjects: Enzymes -- Analysis
Hong Kong Polytechnic University -- Dissertations
Issue Date: 2001
Publisher: The Hong Kong Polytechnic University
Abstract: The objective of this project is to identify the important polypeptide regions in pyridoxal kinase (PK) enzyme which is involved in the activation of vitamin B₆ in many organisms. To achieve this goal, the construction of chimeric enzymes was carried out and different properties of these chimeric enzymes were studied. A library of chimeric genes encoding a family of chimeric PK enzymes could be derived from two partially homologous genes (the porcine PK gene and the human PK gene) by requiring an in vivo recombination event with the selection of recombinant DNAs. The great advantage of this technique is the potential for rapidly generating a large number of chimeric enzymes that can be used to map functional regions of the polypeptide. The recombination machineries of Bacillus subtilis 168 and Escherichia coli RR1 were used and the E. coli system was found to be more efficient in generating the hybrid genes. Chimeric PK genes have been efficiently assembled in E. coli RR1 by intramolecular recombination within homologous segments of the regions encoding the two parent proteins. The recombination efficiency of E. coli RR1 was 83 colonies/μg of linearized DNA. We sequenced 19 hybrids and found that 16 of these were different based on the nucleotide sequences.
Five hybrids were selected for study in this project and they are designated as Hybrid 2, Hybrid 22, Hybrid 26, Hybrid 28 and Hybrid 34. The results of Western blotting experiment indicated that the monoclonal antibody raised against porcine PK could bind to porcine, human and hybrid PK enzymes respectively which were overexpressed and isolated from E. coli cells. This suggested that the structures of the two wild-types and the five selected hybrids should be similar. The far-UV spectra of circular dichroism further suggested that the two purified wild-type enzymes, the purified Hybrid 34 and Hybrid 22 have similar secondary structures. In a 10% non-denaturing polyacrylamide gel, it was found that the two wild-types, Hybrid 34 and Hybrid 22 were all existed in dimer forms. Although there is a high similarity in the primary structure between porcine and human PK enzymes, their expression levels in E. coli BL21(DE3) were very different. The five selected hybrids gave different expression levels in the E. coli system under the T7 promoter regulation. It was observed that the hybrids with a larger proportion derived from the porcine PK had a higher expression level or vice versa. When using the same purification procedure, the two wild-type enzymes and the hybrids displayed different chromatographic properties. Detailed kinetic studies of these hybrid PK enzymes and their parent enzymes suggested that the amino acid residues 32 - 196 of the porcine PK had a large contribution to the substrate pyridoxal (PL) binding. Also, replacement of the amino acid residues 1 - 32 of the porcine PK by those from human had some effect on the substrate adenosine triphosphate (ATP) binding process. The amino acid residues 186-312 of human PK should be important for catalytic activities as the catalytic rate for the formation of pyridoxal phosphate (PLP) was affected drastically by the replacement of this region.
Degree: M.Phil., Dept. of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, 2001
Description: x, 161 leaves : ill. ; 30 cm.
PolyU Library Call No.: [THS] LG51 .H577M ABCT 2001 Kwok
Rights: All rights reserved.
Type: Thesis
URI: http://hdl.handle.net/10397/3301
Appears in Collections:ABCT Theses
PolyU Electronic Theses

Files in This Item:

File Description SizeFormat
b15533591_ir.pdfFor All Users (Non-printable) 19.55 MBAdobe PDFView/Open
b15533591_link.htmFor PolyU Users 161 BHTMLView/Open



Facebook Facebook del.icio.us del.icio.us LinkedIn LinkedIn


All items in the PolyU Institutional Repository are protected by copyright, with all rights reserved, unless otherwise indicated.
No item in the PolyU IR may be reproduced for commercial or resale purposes.

 

© Pao Yue-kong Library, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong
Powered by DSpace (Version 1.5.2)  © MIT and HP
Feedback | Privacy Policy Statement | Copyright & Restrictions - Feedback