Trypanosomes are eukaryotic parasites that can infect a variety of hosts and result in multiple diseases for which therapeutics are lacking. Trypanosoma brucei contains two putative haloacid dehalogenases, or HADs, that were acquired by the parasites from bacteria through horizontal gene transfer. One of these, HAD32, is essential in their life cycle. Both trypanosome HADs are uncharacterized. Exploring the function of HAD32 may hold therapeutic potential. We seek to find the function of the trypanosome HADs, starting with their enzymatic activity. Recombinant His10-TbHAD32 was overexpressed in bacteria and purified to approximate homogeneity. The HAD superfamily of enzymes utilizes a wide range of substrates. TbHAD32 has a sequence motif associated with phosphatase activity within the HAD superfamily. We screened several potential substrates for the protein and found that Trypanosoma brucei HAD32 is a phosphatase. His10-TbHAD32 has a catalytic efficiency of 65.73 +/- 12.44 mM-1 min-1 when utilizing para-nitrophenol phosphate (PNPP) as a substrate. With PNPP as substrate, we determined that Mg2+ is the optimal metal required for His10-TbHAD32 phosphatase activity. With broad activity in hand, we have identified three additional and potentially biologically relevant compounds that His10-TbHAD32 utilizes as substrates and have determined the kinetic parameters of the enzyme with these compounds. These activities give us a solid foundation upon which to build hypotheses of enzyme function in the parasite.
