Thymidylate synthase (TS) converts dUMP to dTMP by reductive methylation, where 5,10-methylenetetrahydrofolate is the source of both the methylene group and reducing equivalents. The mechanism of this reaction has been extensively studied, mainly using the enzyme from Escherichia coli. Bacillus subtilis contains two genes for TSs, ThyA and ThyB. The ThyB enzyme is very similar to other bacterial TSs, but the ThyA enzyme is quite different, both in sequence and activity. In ThyA TS, the active site histidine is replaced by valine. In addition, the B. subtilis enzyme has a 2.4-fold greater kcat than the E. coli enzyme. The structure of B. subtilis thymidylate synthase in a ternary complex with 5-fluoro-dUMP and 5,10-methylenetetrahydrofolate has been determined to 2.5 Å resolution. Overall, the structure of B. subtilis TS (ThyA) is similar to that of the E. coli enzyme. However, there are significant differences in the structures of two loops, the dimer interface and the details of the active site. The effects of the replacement of histidine by valine and a serine to glutamine substitution in the active site area, and the addition of a loop over the carboxy terminus may account for the differences in kcat found between the two enzymes.

β-Galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer. Each subunit consists of five domains, the third being an α/β barrel that contains most of the active site residues. A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank. Many structures include an α/β barrel. Those that are most similar to the α/β barrel of E. coli β-galactosidase have similar catalytic residues and belong to the so-called “4/7 superfamily” of glycosyl hydrolases. The structure comparison suggests that β-amylase should also be included in this family. Of three structure comparison methods tested, the “ProSup” procedure of Zu-Kang and Sippl and the “Superimpose” procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures. Domains 1, 2, and 4 of E. coli β-galactosidase have topologies related to “jelly-roll barrels” and “immunoglobulin constant” domains. This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases. The fold of domain 1 of E. coli β-galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs. This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in β-galactosidase that are unrelated to the functions that such domains provide in other contexts. It is proposed that β-galactosidase arose from a prototypical single domain α/β barrel with an extended active site cleft. The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, allolactose.