Domain-specific characteristics of the bifunctional key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.
UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is a bifunctional enzyme, which initiates and regulates sialic acid biosynthesis. Sialic acids are important compounds of mammalian glycoconjugates, mediating several biological processes, such as cell-cell or cell-matrix interactions. In order to characterize the function of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, a number of deletion mutants were generated, lacking either parts of the N-terminal epimerase or the C-terminal kinase domain. N-terminal deletion of only 39 amino acids results in a complete loss of epimerase activity. Deletions in the C-terminal part result in a reduction or complete loss of kinase activity, depending on the size of the deletion. Deletions at either the N- or the C-terminus also result in a reduction of the other enzyme activity. These results indicate that a separate expression of both domains is possible, but that a strong intramolecular dependency of the two domains has arisen during evolution of the enzyme. N-terminal, as well as C-terminal, mutants tend to form trimers, in addition to the hexameric structure of the native enzyme. These results and yeast two-hybrid experiments show that structures required for dimerization are localized within the kinase domain, and a potential trimerization site is possibly located in a region between the two domains. In conclusion, our results reveal that the activities, as well as the oligomeric structure, of this bifunctional enzyme seem to be organized and regulated in a complex manner.
Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Institut für Biochemie und Molekularbiologie, Arnimallee 22, 14195 Berlin-Dahlem, Germany.
Astrid
A
Blume
Wenke
W
Weidemann
Ulrich
U
Stelzl
Erich E
EE
Wanker
Lothar
L
Lucka
Peter
P
Donner
Werner
W
Reutter
Rüdiger
R
Horstkorte
Stephan
S
Hinderlich
D000818:Animals; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D051381:Rats; D017384:Sequence Deletion / Q000235:genetics; D012794:Sialic Acids / Q000096:biosynthesis* / Q000378:metabolism; D018411:Spodoptera; D020798:Two-Hybrid System Techniques
D012794:Sialic Acids; D017853:Phosphotransferases (Alcohol Group Acceptor); C018765:N-acylmannosamine kinase; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase