Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase (TS) to catalyze the methylation of the uracil moiety of 2’-deoxyuridine-5’-monophosphate (dUMP). All known thymidylate synthases (TSs) rely on an active site residue of the enzyme to activate dUMP1, 2. This functionality has been demonstrated for classical TSs, including human TS, and is instrumental in mechanism-based inhibition of these enzymes. Here we report the first example of thymidylate biosynthesis that occurs without an enzymatic nucleophile. This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens3–5. Our findings indicate that the putative active site nucleophile is not required for FDTS catalysis, and no alternative nucleophilic residues capable of serving this function can be identified. Instead, our findings suggest that a hydride equivalent (i.e. a proton and two electrons) is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2’-deoxythymidine-5’-monophosphate (dTMP). These observations indicate a very different chemical cascade than that of classical TSs or any other known biological methylation. The findings and chemical mechanism proposed here, together with available structural data, suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis, should be feasible. Since several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs.

The two long-known "classical" enzymes of uridyl-5-methylation, thymidylate synthase and ribothymidyl synthase, have been joined by two alternative methylation enzymes, flavin-dependent thymidylate synthase and folate-dependent ribothymidyl synthase. These two newly discovered enzymes have much in common: both contain flavin cofactors, utilize methylenetetrahydrofolate as a source of methyl group, and perform thymidylate synthesis via chemical pathways distinct from those of their classic counterparts. Several severe human pathogens (e.g., typhus, anthrax, tuberculosis, and more) depend on these "alternative" enzymes for reproduction. These and other distinctive properties make the alternative enzymes and their corresponding genes appealing targets for new antibiotics.

New findings lead to a revised understanding of the substrates' binding order, the role of the substrate as an activator, and the observed lag phase in the FDTS catalyzed reaction.

present in microorganisms, plants and animals. From a biotechnological point of view, Kluyveromyces lactis β-galactosidase is suitable for many applications due to its neutral optimum pH and for the fact that K. lactis is a GRAS organism (Generally Recognized As Safe). Interestingly, β-galactosidases are being used in lactose intolerance treatments and in food industry. Moreover, yeast expressing this enzyme can be used to improve the valorization of the cheese whey, a cheese industry byproduct by coupling the degradation of lactose with ethanol production, biomass production, etc. [1]. On the basis of their sequence, β-galactosidases are classified within families 1, 2, 35 and 42 of glycosyl hydrolases in the CAZy database [2]. Those from eukaryotic organisms are grouped into family 35 with the only exceptions of K. lactis and K. marxianus β-galactosidases which belong to family 2, together with the prokaryotic β-galactosidases from Escherichia coli and Arthrobacter sp. Whereas the structures of these last two prokaryotic enzymes have been determined [3], [4], none of the eukaryotic β-galactosidase structures have been reported to date. Although their sequence similarity with the prokaryotic enzymes is significant (48% vs. E. coli and 47% vs. Arthrobacter) there are many differences, particularly some long insertions and deletions, which play an important role in protein stability and in substrate recognition and specificity. Gaining insight into the structural features that determine its stability and understanding the specificity determinants and catalytic mechanism should lead to improvements of its biotechnological applications by rational protein engineering. In this study, we describe X-ray crystallographic studies and an analysis of Kluyveromyces lactis β-galactosidase structure.