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<sub>m</sub> 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. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/54205 |
| Date | January 1989 |
| Creators | Jiang, Weiping |
| Contributors | Biochemistry and Nutrition, Niehaus, Walter G. Jr., Anderson, Bruce M., Ferry, James G., Ebel, Richard E., Larson, Timothy J. |
| Publisher | Virginia Polytechnic Institute and State University |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | xii, 135 leaves, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 22354887 |
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