Abstract

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-arabino pyranose (UDP-Arap), which differs from UDP-Galp by lacking a -CH 2 -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 -CH 2 -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.

Department(s)

Chemistry

Comments

National Science Foundation, Grant CHE-1506206

International Standard Serial Number (ISSN)

1520-4995; 0006-2960

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2024 American Chemical Society, All rights reserved.

Publication Date

03 Jul 2018

PubMed ID

29757624

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