Cloning and characterization of glucokinase from a methylotrophic yeast Hansenula polymorpha: different effects on glucose repression in H. polymorpha and Saccharomyces cerevisiae.
Glucokinase gene (HPGLK1) was cloned from a methylotrophic yeast Hansenula polymorpha by complementation of glucose-phosphorylation deficiency in a H. polymorpha double kinase-negative mutant A31-10 by a genomic library. An open reading frame of 1416 nt encoding a 471-amino-acid protein with calculated molecular weight 51.6 kDa was characterized in the genomic insert of the plasmid pH3. The protein sequence deduced from HPGLK1 exhibited 55 and 46% identity with glucokinases from Saccharomyces cerevisiae and Aspergillus niger, respectively. The enzyme phosphorylated glucose, mannose and 2-deoxyglucose, but not fructose. Transformation of HPGLK1 into A31-10 restored glucose repression of alcohol oxidase and catalase in the mutant. Transformation of HPGLK1 into S. cerevisiae triple kinase-negative mutant DFY632 showed that H. polymorpha glucokinase cannot transmit the glucose repression signal in S. CEREVSIAE: synthesis of invertase and maltase in respective transformants was insensitive to glucose repression similarly to S. cerevisiae DFY568 possessing only glucokinase.
Laht
Silja
S
Institute of Molecular and Cell Biology, University of Tartu, Riia 23, Estonia.
Karp
Helen
H
Kotka
Pille
P
Järviste
Aiki
A
Alamäe
Tiina
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D015698:Genomic Library; D005941:Glucokinase / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010802:Phylogeny; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005941:Glucokinase; D005947:Glucose