The trehalose biosynthetic pathway is of great interest for the development of novel therapeutics, since trehalose is an essential disaccharide in many pathogens, but is neither required nor synthesised in mammalian hosts. As such, trehalose-6-phosphate phosphatase (TPP), a key enzyme in trehalose biosynthesis is an attractive target for novel chemotherapeutics. Based on a survey of genomes from a panel of parasitic nematodes and bacterial organisms, and by way of a structure-based amino acid sequence alignment, we derive the topological structure of mono-enzyme trehalose-6-phosphate phosphatases and classify them into three groups. Comparison of the functional roles of amino acid residues located in the active site of TPPs belonging to different groups reveals nuanced variation. Since current literature on this enzyme family shows a tendency to infer functional roles for individual amino acid residues, we investigated the roles of the strictly conserved aspartate tetrad in TPP of the nematode Brugia malayi by using a conservative mutation approach. In contrast to aspartate-213, the residue inferred to carry out the nucleophilic attack on the substrate, we found that aspartate-215 and aspartate-428 of BmTPP are involved in the chemistry steps of enzymatic hydrolysis of the substrate. We therefore suggest that homology-based inference of functionally important amino acids by sequence comparison for mono-enzyme TPPs should only be carried out within each of the three groups.