Rhamnose-associated molecules are attracting attention because they are present in bacteria but not mammals, making them potentially useful as antibacterial agents. Additionally, they are also valuable for tumor immunotherapy. Thus, studies on the functions and biosynthetic pathways of rhamnose-containing compounds are in progress. In this paper, studies on the biosynthetic pathways of three rhamnose donors, i.e., deoxythymidinediphosphate-L-rhamnose (dTDP-Rha), uridine diphosphate-rhamnose (UDP-Rha), and guanosine diphosphate rhamnose (GDP-Rha), are firstly reviewed, together with the functions and crystal structures of those associated enzymes. Among them, dTDP-Rha is the most common rhamnose donor, and four enzymes, including glucose-1-phosphate thymidylyltransferase RmlA, dTDP-Glc-4,6-dehydratase RmlB, dTDP-4-keto-6-deoxy-Glc-3,5-epimerase RmlC, and dTDP-4-keto-Rha reductase RmlD, are involved in its biosynthesis. Secondly, several known rhamnosyltransferases from Geobacillus stearothermophilus, Saccharopolyspora spinosa, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Streptococcus pneumoniae are discussed. In these studies, however, the functions of rhamnosyltransferases were verified by employing gene knockout and radiolabeled substrates, which were almost impossible to obtain and characterize the products of enzymatic reactions. Finally, the application of rhamnose-containing compounds in disease treatments is briefly described.

A new, robust one-pot four-enzyme synthetic method was developed for thymidinediphosphate-L-rhamnose starting from D-glucose-1-phosphate. The enzymes, Glc-1-P thymidylyltransferase, dTDP-Glc-4,6-dehydratase, dTDP-4-keto-6-deoxy-Glc-3,5-epimerase and dTDP-4-keto-Rha reductase were derived from Streptococcus pneumonia serotype 23F, expressed in Escherichia coli, and studied in detail to provide the first direct evidence for their functions.

A new β-rhamnoslytransferase Cps23FT from Streptococcus pneumonia serotype 23F was expressed and characterized. Its enzymatic activity and function were confirmed for the first time by utilizing enzymatically prepared dTDP-Rha and chemically synthesized Glcα-PP-(CH₂)₁₁-OPh as substrates. This reaction gave the desired disaccharide Rhaβ-1,4-Glcα-PP-(CH₂)₁₁-OPh in a good isolated yield (67%), suggesting the potential of Cps23FT as a tool enzyme for the synthesis of complex oligosaccharides containing difficult β-rhamnosyl linkages. Furthermore, site-directed mutagenesis of Cps23FT disclosed that its ²⁷¹DKD²⁷³ motif was critical for the enzymatic activity and most likely the binding site for the required divalent metal cation.