Rodolpho Do Aido-Machado

Flap endonuclease (FEN) enzymes are a group of metallonucleases that have essential roles in DNA repair and the maintenance of genomic resilience. They catalyse hydrolytic cleavage of a phosphodiester bond to remove 5′-flaps present on double-stranded DNA molecules formed during DNA replication. FEN locates a target scissile bond through the structural recognition of a pronounced bend in the double-stranded DNA substrate along with the presence of both a 5′-flap and a 1-nucleotide 3′-flap. FEN enzymes share a common structural architecture and are functionally conserved across all living organisms. In this work, we report the ¹H, ¹⁵N and ¹³C backbone resonance assignments of residues 2–349 of FEN from Plasmodium falciparum (PfFEN349) in its substrate-free state. Using heteronuclear multidimensional NMR spectroscopy, 90% of all backbone resonances of PfFEN349 were assigned, with 298 backbone amide resonances out of 337 theoretically-detectible resonances (which exclude 10 prolines and the N-terminal glycine) identified in the ¹H–¹⁵ N TROSY spectrum. Prediction of solution secondary structure content from a chemical shift analysis using the TALOS-N webserver is mostly in good agreement with an AlphaFold model of PfFEN349. The reported assignments provide a pathway for drug discovery as PfFEN349 is a potential target for the development of new inhibitors that could be utilised to control the incidence of malaria across the globe.

Addition of glycine betaine up to 1 M gave rise to increased intensity for some weak signals in the HSQC spectra of barnase and Plasmodium falciparum flap endonuclease. The signals that were enhanced were low intensity signals, often from amide groups with rapid internal motion (low order parameter). The majority of signals are however somewhat weaker because of the increased viscosity. Addition of betaine is shown to cause a small but significant overall increase in order parameter, no consistent effect on conformational change on the µs-ms timescale, and a reduction in amide exchange rates, by a factor of about 3. The results are consistent with a model in which betaine leads to a reduction in fluctuations within the bulk water, which in turn produces a reduction in internal fluctuations of the protein.

Selective oxyfunctionalization of non-activated C–H bonds remains a major challenge in synthetic chemistry. The biocatalytic hydroxylation of non-activated C–H bonds by cytochrome P450 monooxygenases (CYPs), however, offers catalysis with high regio- and stereoselectivity using molecular oxygen. CYP153s are a class of CYPs known for their selective terminal hydroxylation of n-alkanes and microorganisms, such as the bacterium Alcanivorax dieselolei, have evolved extensive enzymatic pathways for the oxyfunctionalization of various lengths of n-alkanes, including a CYP153 to yield medium-chain 1-alkanols. In this study, we report the characterization of the terminal alkane hydroxylase from A. dieselolei (CYP153A71) for the oxyfunctionalization of medium-chain n-alkanes in comparison to the well-known CYP153A6 and CYP153A13. Although the expected 1-alkanols are produced, CYP153A71 readily converts the 1-alkanols to the corresponding aldehydes, fatty acids, as well as α,ω-diols. CYP153A71 is also shown to readily hydroxylate medium-chain fatty acids. The X-ray crystal structure of CYP153A71 bound to octanoic acid is solved, yielding an insight into not only the regioselectivity, but also the binding orientation of the substrate, which can be used in future studies to evolve CYP153A71 for improved oxidations beyond terminal n-alkane hydroxylation.

Baeyer-Villiger monooxygenases (BVMOs) are flavin-dependent enzymes that primarily convert ketones to esters, but can also catalyze heteroatom oxidation. Several structural studies have highlighted the importance of the ‘control loop’ in BVMOs, which adopts different conformations during catalysis. Central to the ‘control loop’ is a conserved tryptophan that has been implicated in NADP(H) binding. BVMO_AFL210 from Aspergillus flavus, however, contains a threonine in the equivalent position. Here, we report the structure of BVMO_AFL210 in complex with NADP⁺ in both the ‘open’ and ‘closed’ conformations. In neither conformation does Thr513 contact the NADP⁺. Although mutagenesis of Thr513 did not significantly alter the substrate scope, changes in peroxyflavin stability and reaction rates were observed. Mutation of this position also brought about changes in the regio- and enantioselectivity of the enzyme. Moreover, lower rates of overoxidation during sulfoxidation of thioanisole were also observed.

TbFKBP12 is a putative peptidyl prolyl cis–trans isomerase from Trypanosoma brucei, causative agent of the African trypanosomiasis or sleeping sickness. It interacts with the immunosuppressive drug rapamycin inhibiting the formation of TORC2 complex leading to parasite death by inhibiting cell proliferation through cytokinesis blockade. Moreover, RNAi silencing of TbFKBP12 revealed essential function in both procyclic and bloodstream forms. Both facts make TbFKBP12 an attractive target for ligand development and thus structural data is desirable. In this work we report the NMR resonance assignments for ¹H, ¹⁵N and ¹³C nuclei in the backbone and side chains of the TbFKBP12 as basis for further studies of structure, backbone dynamics, interaction mapping and drug screening.