posted on 2020-08-22, 00:13authored byPavel Janoš, Igor Tvaroška, Christoph Dellago, Jaroslav Koča
We
applied the transition path sampling (TPS) method to study the
translocation step of the catalytic mechanism of galactofuranosyl
transferase 2 (GlfT2). Using TPS in the field of enzymatic reactions
is still relatively rare, and we show its effectiveness on this enzymatic
system. We decipher an unknown mechanism of the translocation step
and, thus, provide a complete understanding of the catalytic mechanism
of GlfT2 at the atomistic level. The GlfT2 enzyme is involved in the
formation of the mycobacterial cell wall and transfers galactofuranose
(Galf) from UDP-Galf onto a growing acceptor Galf chain. The biosynthesis
of the galactan chain is accomplished in a processive manner, with
the growing acceptor substrate remaining bound to GlfT2. The glycosidic
bond formed by GlfT2 between the two Galf residues alternates between
β-(1–6) and β-(1–5) linkages. The translocation
of the growing galactan between individual additions of Galf residues
is crucial for the function of GlfT2. Analysis of unbiased trajectory
ensembles revealed that the translocation proceeds differently depending
on the glycosidic linkage between the last two Galf residues. We also
showed that the protonation state of the catalytic residue Asp372
significantly influences the translocation. Approximate transition
state structures and potential energy reaction barriers of the translocation
process were determined. The calculated potential reaction barriers
in the range of 6–14 kcal/mol show that the translocation process
is not the rate-limiting step in galactan biosynthesis.