A single amino-acid substitution toggles chloride dependence of the alpha-amylase paralog amyrel in Drosophila melanogaster and Drosophila virilis species.
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.
UMR 9191 Evolution, Génomes, Comportement et Ecologie, CNRS, F-91198 Gif-sur-Yvette, France; Univ. Paris-Sud, F-91405 Orsay Cedex, France.
Gaëlle
G
Claisse
Laboratory of Biochemistry, Center for Protein Engineering, University of Liège, B-4000 Liège-Sart Tilman, Belgium.
Georges
G
Feller
UMR 9191 Evolution, Génomes, Comportement et Ecologie, CNRS, F-91198 Gif-sur-Yvette, France; Univ. Paris-Sud, F-91405 Orsay Cedex, France.
Magalie
M
Bonneau
UMR 9191 Evolution, Génomes, Comportement et Ecologie, CNRS, F-91198 Gif-sur-Yvette, France; Univ. Paris-Sud, F-91405 Orsay Cedex, France. Electronic address: jean-luc.da-lage@egce.cnrs-gif.fr.
Jean-Luc
JL
Da Lage
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000681:Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002712:Chlorides / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004330:Drosophila / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D002712:Chlorides; D018076:DNA, Complementary; D029721:Drosophila Proteins; D012333:RNA, Messenger; D000681:Amylases; C112911:Amyrel protein, Drosophila; D000516:alpha-Amylases
Chloride-dependent enzyme; Digestive enzyme; Drosophila; Protein evolution; α-Amylase