Archives of microbiology vol:167 issue:4 pages:202-208
We cloned the Kluyveromyces lactis KINTH1 gene, which encodes neutral trehalase. It showed 65.2% and 68.5% identity at nucleotide and amino acid sequence level, respectively, with the Saccharomyces cerevisiae NTH1 gene. Multiple alignment of the predicted trehalase protein sequences from yeasts, bacteria, insects, and mammals revealed two major domains of conservation. Only the yeast trehalases displayed in an N-terminal extension two consensus sites for cAMP-dependent protein phosphorylation and a putative Ca2+-binding sequence. Gene disruption of the KINTH1 gene abolished neutral trehalase activity and clearly revealed a trehalase activity with an acid pH optimum. It also resulted In a high constitutive trehalose level. Expression of the KINTH1 gene in an S. cerevisiae nth1 Delta mutant resulted in rapid activation of the heterologous trehalase upon addition of glucose to cells growing on a nonfermentable carbon source and upon addition elf a nitrogen source to cells starved for nitrogen in a glucose-containing medium. In ii. lactis, the same responses were observed except that rapid activation by glucose was observed only in early-exponential-phase cells. Inactivation of K. lactis neutral trehalase by alkaline phosphatase and activation by cAMP in cell extracts are consistent with control of the enzyme by cAMP-dependent protein phosphorylation.