The L-Rhamnose Biosynthetic Pathway in Trichomonas vaginalis: Identification and Characterization of UDP-D-Glucose 4,6-dehydratase

Abstract

Trichomonas vaginalis is the causative agent of one of the most widespread sexually transmitted diseases in the world. The adhesion of the parasite to the vaginal epithelial cells is mediated by specific proteins and by a complex glycan structure, the lipoglycan (TvLG), which covers the pathogen surface. L-rhamnose is an important component of TvLG, comprising up to 40% of the monosaccharides. Thus, the inhibition of its production could lead to a severe alteration in the TvLG structure, making the L-rhamnose biosynthetic pathway an attractive pharmacologic target. We report the identification and characterization of the first committed and limiting step of the L-rhamnose biosynthetic pathway, UDP-D-glucose 4,6-dehydratase (UGD, EC 4.2.1.76). The enzyme shows a strong preference for UDP-D-glucose compared to dTDP-D-glucose; we propose that the mechanism underlying the higher affinity for the UDP-bound substrate is mediated by the differential recognition of ribose versus the deoxyribose of the nucleotide moiety. The identification of the enzymes responsible for the following steps of the L-rhamnose pathway (epimerization and reduction) was more elusive. However, sequence analyses suggest that in T. vaginalis L-rhamnose synthesis proceeds through a mechanism different from the typical eukaryotic pathways, displaying intermediate features between the eukaryotic and prokaryotic pathways and involving separate enzymes for the epimerase and reductase activities, as observed in bacteria. Altogether, these results form the basis for a better understanding of the formation of the complex glycan structures on TvLG and the possible use of L-rhamnose biosynthetic enzymes for the development of selective inhibitors.

Arabidopsis thaliana RHAMNOSE 1 condensate formation drives UDP-rhamnose synthesis

“…S7C), suggesting rffG may form condensates. Similarly to E.coli, the protozoan T. vaginalis synthesizes rhamnose for their lipoglycan outer surface which is an important virulence factor (31). Trichomonas vaginalis NDP-D-Glucose 4,6dehydratase forms homodimers (31) and is similar in protein sequence to RHM1 BD1.…”

“…Similarly to E.coli, the protozoan T. vaginalis synthesizes rhamnose for their lipoglycan outer surface which is an important virulence factor (31). Trichomonas vaginalis NDP-D-Glucose 4,6dehydratase forms homodimers (31) and is similar in protein sequence to RHM1 BD1. In the multicellular worm C. elegans, rhamnose is required for embryogenesis and larval molting, likely by forming a component of the outer cuticle (32).…”

“…How deeply are rhamnosomes conserved and when did they evolve? Outside of plants, rhamnose is synthesized in bacteria ( 28, 29 ), fungi ( 30 ), protozoa ( 31 ), and some invertebrate animals ( 32 ). RHM1 homologs (composed of both BD1 and BD2) are present in unicellular algae, mosses, and angiosperms (Fig.…”

“…S7A and B), suggesting rhamnosomes are formed throughout the plant kingdom. In prokaryotes and non-plant eukaryotes that synthesize rhamnose, such as Escherichia coli, Trichomonas vaginalis , and Caenorhabditis elegans , nucleotide diphosphate (NDP)-rhamnose synthesis typically requires three monofunctional enzymes ( 23, 31, 32 ). E. coli requires rhamnose for synthesis of glycan used for the lipopolysaccharide in the cell wall ( 33 ) and the first monofunctional enzyme in rhamnose synthesis is a member of the NAD(P)-binding domain superfamily (dTDP-glucose 4,6-dehydratase 2; rffG) ( 34 ).…”

“…Protein sequences for RHM1-homologs from viridiplantae proteins were identified using NCBI blast. Protein sequences from bacteria ( 28, 29 ), fungi ( 30 ), protozoa ( 31 ), and invertebrate animals ( 32 ) were identified from previous literature, and their amino acid sequences were retrieved from Uniprot. Protein sequences used for alignment and phylogenetic tree construction are in Table S5.…”