Isolation and characterization of glutaminyl cyclases from Drosophila: evidence for enzyme forms with different subcellular localization.
Glutaminyl cyclases (QCs) present in plants and vertebrates catalyze the formation of pyroglutamic acid (pGlu) from N-terminal glutamine. Pyroglutamyl hormones also identified in invertebrates imply the involvement of QC activity during their posttranslational maturation. Database mining led to the identification of two genes in Drosophila, which putatively encode QCs, CG32412 (DromeQC) and CG5976 (isoDromeQC). Analysis of their primary structure suggests different subcellular localizations. While DromeQC appeared to be secreted due to an N-terminal signal peptide, isoDromeQC contains either an N-terminal mitochondrial targeting or a secretion signal due to generation of different transcripts from gene CG5976. According to the prediction, homologous expression of the corresponding cDNAs in S2 cells revealed either secreted protein in the medium or intracellular QC activity. Subcellular fractionation and immunochemistry support export of isoDromeQC into the mitochondrion. For enzymatic characterization, DromeQC and isoDromeQC were expressed heterologously in Pichia pastoris and Escherichia coli, respectively. Compared to mammalian QCs, the specificity constants were about 1 order of magnitude lower for most of the analyzed substrates. The pH dependence of the specificity constant was similar for both enzymes, indicating the necessity of an unprotonated substrate amino group and two protonated groups of the enzyme, resulting in an asymmetric bell-shaped characteristic. The determination of the metal content of DromeQC revealed equimolar protein-bound zinc. These results prove conserved enzymatic mechanisms between QCs from invertebrates and mammals. Drosophila is the first organism for which isoenzymes of glutaminyl cyclase have been isolated. The identification of a mitochondrial QC points toward yet undiscovered physiological functions of these enzymes.
Schilling
Stephan
S
Probiodrug AG, Weinbergweg 22, 06120 Halle/Saale, Germany.
Lindner
Christiane
C
Koch
Birgit
B
Wermann
Michael
M
Rahfeld
Jens-Ulrich
JU
von Bohlen
Alex
A
Rudolph
Thomas
T
Reuter
Gunter
G
Demuth
Hans-Ulrich
HU
D000595:Amino Acid Sequence / Q000235:genetics; D019881:Aminoacyltransferases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D004330:Drosophila / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000201:enzymology*; D013379:Substrate Specificity / Q000235:genetics
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase; D011761:Pyrrolidonecarboxylic Acid