A single amino-acid substitution toggles chloride dependence of the alpha-amylase paralog amyrel in Drosophila melanogaster and Drosophila virilis species

• The a-amylase paralogue Amyrel in Drosophila is known from its gene sequence but has not been characterized to date.
• We have produced in yeast, and characterized Amyrel from Drosophila melanogaster and Drosophila virilis.
• In D. melanogaster, Amyrel is chloride-dependent; the mutation R323Q suppresses chloride activation and decreases activity.
• In D. virilis, Amyrel is chloride-independent; chloride activation was enabled by the mutation Q323Rwith increased.
• The results highlight the function of the chloride ion as an allosteric activator of activity in animal amylolytic enzymes.

In animals, most α-amylases are chloride-dependent enzymes. A chloride ion is required for allosteric activation and is coordinated by one asparagine and two arginine side chains. Whereas the asparagine and one arginine are strictly conserved, the main chloride binding arginine is replaced by a glutamine in some rare instances, resulting in the loss of chloride binding and activation. Amyrel is a distant paralogue of α-amylase in Diptera, which was not characterized biochemically to date. Amyrel shows both substitutions depending on the species. In Drosophila melanogaster, an arginine is present in the sequence but in Drosophila virilis, a glutamine occurs at this position. We have investigated basic enzymological parameters and the dependence to chloride of Amyrel of both species, produced in yeast, and in mutants substituting arginine to glutamine or glutamine to arginine. We found that the amylolytic activity of Amyrel is about thirty times weaker than the classical Drosophila α-amylase, and that the substitution of the arginine by a glutamine in D. melanogaster suppressed the chloride-dependence but was detrimental to activity. In contrast, changing the glutamine into an arginine rendered D. virilis Amyrel chloride-dependent, and interestingly, significantly increased its catalytic efficiency. These results show that the chloride ion is not mandatory for Amyrel but stimulates the reaction rate. The possible phylogenetic origin of the arginine/glutamine substitution is also discussed.

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