posted on 2018-05-14, 00:00authored byGustavo Pierdominici-Sottile, Rodrigo Cossio-Pérez, Isabel Da Fonseca, Karina Kizjakina, John J. Tanner, Pablo Sobrado
Galactose is an abundant
monosaccharide found exclusively in mammals
as galactopyranose (Galp), the six-membered ring
form of this sugar. In contrast, galactose appears in many pathogenic
microorganisms as the five-membered ring form, galactofuranose (Galf). Galf biosynthesis begins with the conversion
of UDP-Galp to UDP-Galf catalyzed
by the flavoenzyme UDP-galactopyranose mutase (UGM). Because UGM is
essential for the survival and proliferation of several pathogens,
there is interest in understanding the catalytic mechanism to aid
inhibitor development. Herein, we have used kinetic measurements and
molecular dynamics simulations to explore the features of UGM that
control the rate-limiting step (RLS). We show that UGM from the pathogenic
fungus Aspergillus fumigatus also catalyzes the isomerization
of UDP-arabinopyranose (UDP-Arap), which differs
from UDP-Galp by lacking a -CH2-OH substituent
at the C5 position of the hexose ring. Unexpectedly, the RLS changed
from a chemical step for the natural substrate to product release
with UDP-Arap. This result implicated residues that
contact the -CH2-OH of UDP-Galp in controlling
the mechanistic path. The mutation of one of these residues, Trp315,
to Ala changed the RLS of the natural substrate to product release,
similar to the wild-type enzyme with UDP-Arap. Molecular
dynamics simulations suggest that steric complementarity in the Michaelis
complex is responsible for this distinct behavior. These results provide
new insight into the UGM mechanism and, more generally, how steric
factors in the enzyme active site control the free energy barriers
along the reaction path.