Expression, sequencing, and characterization of mannitol-1- phosphate dehydrogenase genes from Aspergillus parasiticus and Escherichia coli

The genes coding for mannitol-1-phosphate dehydrogenases (mtlD) from Aspergillus parasiticus and Eschericia coli were cloned and sequenced. The two coding regions were highly homologous and the identity was 88.7% at the amino acid level and 81.6% at the nucleotide level. The two genes translate into polypeptides of equal numbers (382) of amino acids with M, of 40,880 and 41,221, respectively. The possible NAD binding sites were identified for both enzymes in the N-terminal regions according to the consensus sequence fingerprint. The C-terminal regions of both enzymes were similar in sequence to the kinase domain of human liver or -rat liver fructose-6-phosphate-2- kinase:fructose·2,6-bisphosphatase, suggesting that the C-terminal regions are involved in fructose-6-phosphate binding. This conclusion was further supported by site-specitic mutagenesis experiments near the 3’ end of the A. parasiticus gene. A modified A. parasiticus mtlD gene directed the expression, in E. coli, of an enzyme in which amino acid residues 362-369 were altered and amino acid residues 370-382 were deleted with respect to the wild type enzyme. This enzyme exhibited 15% of wild type activity with a 3-fold increase in K_m for fructose-6-phosphate. In the 5’ upstream region of the A. parasiticus mtlD gene, no sequence was found which is similar to the consensus sequences derived for either procaryotic or higher eucaryotic gene promoters; however, inverted repeats were identified, which may be important for regulation of gene expression. A sequence similar to the Shine-Dalgarno sequence was found preceding the translation start codon of the A. parasiticus mtlD gene, which is important for its expression in E. coli. In the 3’ downstream region of the A. parasiticus mtlD gene, an additional open reading frame was found, which translated into a polypeptide of 153 amino acids with M, of 17,111. This polypeptide was identified using maxicell experiments.

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biochemistry and Nutrition

Department dc:contributor.department

Biochemistry and Nutrition

Grantor dc:publisher

Virginia Polytechnic Institute and State University

Year dc:date.issued

1989

Author dc:creator

• Jiang, Weiping

Chair dc:contributor.committeechair

• Niehaus, Walter G. Jr.

Committee members dc:contributor.committeemember

• Anderson, Bruce M.
• Ferry, James G.
• Ebel, Richard E.
• Larson, Timothy J.

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en_US

Handle dc:identifier.uri

http://hdl.handle.net/10919/54205

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/54205