Document Detail


Cloning and biochemical characterization of hexokinase from the methylotrophic yeast Hansenula polymorpha.
MedLine Citation:
PMID:  14530868     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We previously showed that, unlike other yeasts, Hansenula polymorpha possesses a glucokinase HPGLK1 that can mediate glucose repression in this yeast, although it cannot replace the regulatory function of hexokinase 2 in Saccharomyces cerevisiae. In the present study, the H. polymorpha hexokinase gene HPHXK1 was cloned by complementation of the glucose growth deficiency of the H. polymorpha double kinase-negative mutant A31-10 with a genomic library. The sequence of the 483-amino acid hexokinase protein deduced from the HPHXK1 gene showed the highest degree of identity (56%) with hexokinase from Schwanniomyces occidentalis, whereas the identity with hexokinase from Kluyveromyces lactis and both hexokinases from Sac. cerevisiae was 55%. The hexokinase protein was purified from crude extracts of H. polymorpha, using ion exchange chromatography and gel filtration. The K(m) values of the purified enzyme for glucose, fructose and ATP were 0.26 mM, 1.1 mM and 0.32 mM, respectively. H. polymorpha hexokinase was inhibited by trehalose-6-phosphate ( K(i)=12 microM) and ADP ( K(i)=1.6 mM), but not by glucose-6-phosphate. Transformation of a H. polymorpha hexokinase-negative mutant with a plasmid carrying the HPHXK1 gene restored the ability of the mutant to phosphorylate fructose and to repress the synthesis of alcohol oxidase and catalase by fructose. Therefore, hexokinase is specifically needed for the establishment of fructose repression in H. polymorpha.
Authors:
Helen Karp; Aiki Järviste; Thomas M Kriegel; Tiina Alamäe
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2003-10-03
Journal Detail:
Title:  Current genetics     Volume:  44     ISSN:  0172-8083     ISO Abbreviation:  Curr. Genet.     Publication Date:  2003 Dec 
Date Detail:
Created Date:  2003-12-08     Completed Date:  2004-07-22     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8004904     Medline TA:  Curr Genet     Country:  United States    
Other Details:
Languages:  eng     Pagination:  268-76     Citation Subset:  IM    
Affiliation:
Institute of Molecular and Cell Biology, Department of Genetics, University of Tartu, Riia 23, 51010 Tartu, Estonia.
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MeSH Terms
Descriptor/Qualifier:
Base Sequence
Cloning, Molecular
Enzyme Inhibitors / pharmacology
Fructose / metabolism
Fungal Proteins / genetics*,  metabolism,  physiology*
Genes, Fungal
Glucose / metabolism
Hexokinase / genetics*,  metabolism,  physiology*
Molecular Sequence Data
Phylogeny
Pichia / enzymology*,  metabolism
Sequence Alignment
Substrate Specificity
Chemical
Reg. No./Substance:
0/Enzyme Inhibitors; 0/Fungal Proteins; 30237-26-4/Fructose; 50-99-7/Glucose; EC 2.7.1.1/Hexokinase

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