The mosquitoes of the Anopheles and Aedes genus are some of the most deadly insects to humans because of their effectiveness as vectors of malaria and a range of arboviruses, including yellow fever, dengue, chikungunya, West Nile and Zika. The use of insecticides from different chemical classes is a key component of the integrated strategy against An. gambiae and Ae. aegypti , but the problem of insecticide resistance means that new compounds with different modes of action are urgently needed to replace chemicals that fail to control resistant mosquito populations. We have previously shown that feeding inhibitors of peptidyl dipeptidase A to both An. gambiae and Ae. aegypti mosquito larvae lead to stunted growth and mortality. However, these compounds were designed to inhibit the mammalian form of the enzyme (angiotensin-converting enzyme, ACE) and hence can have lower potency and lack selectivity as inhibitors of the insect peptidase. Thus, for the development of inhibitors of practical value in killing mosquito larvae, it is important to design new compounds that are both potent and highly selective. Here, we report the first structures of AnoACE2 from An. gambiae in its native form and with a bound human ACE inhibitor fosinoprilat. A comparison of these structures with human ACE (sACE) and an insect ACE homologue from Drosophila melanogaster (AnCE) revealed that the AnoACE2 structure is more similar to AnCE. In addition, important elements that differ in these structures provide information that could potentially be utilised in the design of chemical leads for selective mosquitocide development.

Angiotensin-converting enzyme (ACE) is a zinc metalloprotease best known for its role in blood pressure regulation. ACE consists of two homologous catalytic domains, the N- and C-domain, that display distinct but overlapping catalytic functions in vivo owing to subtle differences in substrate specificity. While current generation ACE inhibitors target both ACE domains, domain-selective ACE inhibitors may be clinically advantageous, either reducing side effects or having utility in new indications. Here, we used site-directed mutagenesis, an ACE chimera and X-ray crystallography to unveil the molecular basis for C-domain-selective ACE inhibition by the bradykinin-potentiating peptide b (BPPb), naturally present in Brazilian pit viper venom. We present the BPPb N-domain structure in comparison with the previously reported BPPb C-domain structure and highlight key differences in peptide interactions with the S 4 to S 9 subsites. This suggests the involvement of these subsites in conferring C-domain-selective BPPb binding, in agreement with the mutagenesis results where unique residues governing differences in active site exposure, lid structure and dynamics between the two domains were the major drivers for C-domain-selective BPPb binding. Mere disruption of BPPb interactions with unique S 2 and S 4 subsite residues, which synergistically assist in BPPb binding, was insufficient to abolish C-domain selectivity. The combination of unique S 9 –S 4 and S 2 ′ subsite C-domain residues was required for the favourable entry, orientation and thus, selective binding of the peptide. This emphasizes the need to consider factors other than direct protein–inhibitor interactions to guide the design of domain-selective ACE inhibitors, especially in the case of larger peptides.

The requirements for substrate binding in the quinoprotein glucose dehydrogenase (GDH) in the membranes of Escherichia coli are described, together with the changes in activity in a site-directed mutant in which His 262 has been altered to a tyrosine residue (H262Y-GDH). The differences in catalytic efficiency between substrates are mainly related to differences in their affinity for the enzyme. Remarkably, it appears that, if a hexose is able to bind in the active site, then it is also oxidized, whereas some pentoses are able to bind (and act as competitive inhibitors), but are not substrates. The activation energies for the oxidation of hexoses and pentoses are almost identical. In a previously published model of the enzyme, His 262 is at the entrance to the active site and appears to be important in holding the prosthetic group pyrroloquinoline quinone (PQQ) in place, and it has been suggested that it might play a role in electron transfer from the reduced PQQ to the ubiquinone in the membrane. The H262Y-GDH has a greatly diminished catalytic efficiency for all substrates, which is mainly due to a marked decrease in their affinities for the enzyme, but the rate of electron transfer to oxygen is unaffected. During the processing of the PQQ into the apoenzyme to give active enzyme, its affinity is markedly dependent on the pH, four groups with p K values between pH 7 and pH 8 being involved. Identical results were obtained with H262Y-GDH, showing that His 262 it is not directly involved in this process.