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Problem Set 5 Key

Lastly other enzymes including weca tago wbco wbpl and

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Unformatted text preview: . Both of their carboxyl groups interact with the amido group of the acceptor asparagine and coordinate Mg2+. These interactions would not be possible if a glutamine or aspartate was present instead of the acceptor asparagine, and provide the molecular basis explaining why glutamines and aspartates are not glycosylated. Additionally, mutating these residues show >90% reduction in PglB activity, providing biochemical evidence that these residues are essential for PglB function. 6 iv. One of the questions regarding N- linked glycosylation is how the acceptor Asn residue is activated for nucleophilic attack of the lipid- linked oligosaccharide. What mechanism do the authors propose that involve the residues discussed in question (iii)? What experimental evidence do the authors provide for their arguments? Do you agree or disagree with their conclusion? Justify your argument. (10 points) The authors propose that residues D56 and E319 are optimally positioned to form hydrogen bonds with the two amide protons of the acceptor asparagine. Forming these hydrogen bonds would require a rotation of the C- N bond of the amido group, which would activate the amide nitrogen for nucleophilic attack. The authors estimate it would require ~18kcal mol- 1 to rotate this bond, and that this energy could be provided by hydrogen bonds from the carboxyl groups of D56 and E319. Mutating these residues to asparagine, glutamine, or alanine almost completely abolishes PglB activity, providing experimental evidence that the carboxyl groups are essential for catalysis. 4. Glycobiology of Bacteria (10 points): a. Chung, B. C., et al. (2013, Science, 341, 1012- 1016) recently published a 3.3Å crystal structure of MraY (phosphor- MurNAc- pentapeptide translocase) from Aquifex aeolicus, which catalyzes an essential step of bacterial peptidoglycan biosynthesis. i. Why has MraY been known to be a promising target for antibiotics? Consider inhibitors and other enzymes in the same superfamily MraY belongs to. (3 points) One reason is because peptidoglycan whose synthesis is catalyzed by MraY is essential in composing of the cell wall of both Gram- ne...
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