NtdB is an enzyme encoded within the ntd operon in Bacillus subtilis. This operon is reported to contain a complete set of genes necessary for the biosynthesis of 3,3'-neotrehalosadiamine (NTD), a compound composed of two kanosamine subunits linked together by a 1,1'-(α,β)-linkage. Both NTD and kanosamine have reported antibiotic properties. The function of NtdB has been a matter of speculation, but has never been investigated in vitro. Using a phosphate assay and an array of substrates, NtdB was determined to be a phosphatase, specific to kanosamine-6-phosphate (K6P) (kcat = 32 ± 1 s-1, Km = 93 ± 7 µM). Site-directed mutagenesis of amino acid residues in the core and the cap domains of the enzyme identified residues important for the catalytic reaction and substrate specificity. These mutations confirmed the presence of four motifs, characteristic of members of the haloacid dehalogenase (HAD) superfamily, and allowed identification of the substrate binding site of the enzyme. KabB, a homologue of NtdB from Bacillus cereus, showed notably lower activity with K6P than NtdB. This research defines the role of NtdB as a specific K6P phosphatase and challenges the previously reported NTD biosynthesis pathway by demonstrating a novel pathway for the production of the antibiotic kanosamine.

Phosphatases of the haloacid dehalogenase superfamily are one of the cell's tools for dephosphorylation of many diverse endogenous and exogenous compounds. This work is aimed at enzymes Tt82 and cytosolic purine 5'-nucleotidase II (cN-II), two members of this large enzyme superfamily. The Tt82 originates in the hyperthermophilic archaeon Thermococcus thioreducens. Up to date, there is only a small amount of knowledge about properties and biological function of this enzyme. Based on its sequence and structure, it was predicted that the Tt82 should possess a phosphatase catalytic activity. Consequently, potential substrates of the Tt82 were proposed by the molecular docking. In this work, the phosphatase activity of the Tt82 was confirmed together with several of its substrates: AMP, D-glucose 1-phosphate, D-glucose 6-phosphate and p-nitrophenyl phosphate (pNPP). Activity towards AMP and pNPP was then characterized by steady-state kinetics at 37 řC and 60 řC. In consistence with its thermophilic origin, the Tt82 showed markedly higher activity towards both substrates at 60 řC. Nonetheless, the effectivity of the Tt82 catalytic activity towards these substrates was actually very low. This leads to assumption, that the identified substrates are probably not biologically relevant. On the other hand, it is quite...