Replacement of the antifreeze-like domain of human N-acetylneuraminic acid phosphate synthase with the mouse antifreeze-like domain impacts both N-acetylneuraminic acid 9-phosphate synthase and 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activities.
Human NeuNAc-9-P synthase is a two-domain protein with ability to synthesize both NeuNAc-9-P and KDN-9-P. Its mouse counterpart differs by only 20 out of 359 amino acids but does not produce KDN-9-P. By replacing the AFL domain of the human NeuNAc-9-P synthase which accommodates 12 of these differences, with the mouse AFL domain we examined its importance for the secondary KDN-9-P synthetic activity. The chimeric protein retained almost half of the ability of the human enzyme for KDN-9-P synthesis while the NeuNAc-9-P production was reduced to less than 10%. Data from the homology modeling and the effect of divalent ions and temperature on the enzyme activities suggest conformational differences between the human and mouse AFL domains that alter the shape of the cavity accommodating the substrates. Therefore, although the AFL domain itself does not define the ability of the human enzyme for KDN-9-P synthesis, it is important for both activities by aiding optimal positioning of the substrates.
Reaves
Marshall Louis
ML
The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA.
Lopez
Linda Carolyn
LC
Daskalova
Sasha Milcheva
SM
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry* / Q000235:genetics; D003198:Computer Simulation; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature
D021301:Antifreeze Proteins; D011993:Recombinant Fusion Proteins; C018767:N-acylneuraminate-9-phosphate synthase; C120800:2-keto-3-deoxy-glycero-galacto-nononic acid 9-phosphate synthetase; D007652:Oxo-Acid-Lyases