Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.
CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, U.S.A.
Ilya B
IB
Mertsalov
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, U.S.A.
Boris N
BN
Novikov
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, U.S.A.
Hilary
H
Scott
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, U.S.A.
Lawrence
L
Dangott
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, U.S.A. panin@tamu.edu.
Vladislav M
VM
Panin
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D009154:Mutation; D000216:N-Acylneuraminate Cytidylyltransferase / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
D029721:Drosophila Proteins; D001224:Aspartic Acid; D008345:Manganese; D000216:N-Acylneuraminate Cytidylyltransferase; D008274:Magnesium
CMP-sialic acid synthetase; Drosophila glycosylation; enzyme evolution; sialic acid; sialylation