Kelsi R. Hall

Tripartite ATP-independent periplasmic (TRAP) transporters are substrate-binding protein dependent secondary transporters that leverage ion gradients to facilitate substrate translocation across bacterial and archaeal membranes. Although the structure of HiSiaQM─a membrane-embedded TRAP subunit from the human pathogen Haemophilus influenzae ─has recently been determined, its transport mechanism is not fully understood. Given the likely importance of lipid interactions and ion coordination dynamics to the transport cycle, we applied multiscale molecular dynamics simulations to investigate these two complementary aspects of HiSiaQM function. Our simulations validated an experimentally identified lipid-binding site (BS1) and provided insight into its lipid-binding dynamics. BS1 is preferentially occupied by the anionic POPG, which is stabilized through persistent hydrogen bonds involving residues H100, R104, and R181. Additionally, our simulations revealed a novel, less stable lipid-binding site (BS2) that is selectively occupied by POPE. We suggest distinct lipid-binding behaviors for POPG and POPE, with POPG forming more persistent interactions at BS1 and POPE engaging more transiently at BS2; however, these remain to be tested experimentally. Analysis of sodium ion coordination at two known binding sites (Na1 and Na2) revealed contrasting dynamics: Na1 exhibited stable ion retention through backbone carbonyl coordination, while Na2 displayed lower stability due to involvement of the flexible side chain of T561. Geometric tunnel analysis identified possible sodium-exit pathways, supporting a working hypothesis that Na1 may act as a stable anchoring site and Na2 may function as a transient ion-exchange point during the transport cycle. Collectively, our findings provide mechanistic insights into how specific lipid interactions and sodium ion coordination could modulate HiSiaQM transporter function.

Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are exceptionally powerful small redox enzymes that master the controlled generation and productive use of potentially damaging hydroxyl radicals in what is essentially a H ₂ O ₂ -driven peroxygenase reaction. We have used ancestral sequence reconstruction and enzyme resurrection to unravel evolutionary steps leading to this exceptional catalytic ability. Real-time monitoring of copper reoxidation and amino acid radical formation showed evolutionary improvement of both the capacity to avoid futile turnover of H ₂ O ₂ and the ability to scavenge damaging radicals resulting from such turnover through a hole hopping pathway. Through mutational studies of ancestral LPMOs, we show that adoption of an extant-like conformation of residues in the hole hopping pathway yields improvements in redox robustness to near-extant levels. These results show how selective pressure imposed by the need for generating a highly oxidizing intermediate is a key driver of metalloenzyme evolution, involving large parts of the enzyme, well beyond the catalytic center.

Lytic polysaccharide monooxygenases (LPMOs) are important biotechnological tools due to their ability to activate C–H bonds in recalcitrant polysaccharides. To-date, most research has focused on LPMOs from the AA9 and AA10 families, while LPMOs from the AA11 family have not received the same attention since their classification almost a decade ago, despite their wide abundance in fungi. Previous studies have shown that AfAA11B from Aspergillus fumigatus has exceptionally high oxidase activity, low reduction potential and the ability to degrade soluble chitooligomers. To better understand the catalytic capabilities of AfAA11B, its crystal structure was solved, revealing a unique flexible surface loop that mediates activity on soluble substrates, as shown by molecular dynamics simulations and mutagenesis. Mutation of an active site Glu residue to a Gln, Asp or Asn showed that this residue is crucial in controlling the low reduction potential and high oxidase activity of AfAA11B. The impact of these mutations on copper reactivity aligned well with results obtained for an AA9 LPMO, which naturally has a Gln in this position. However, the impact of these mutations on the productive peroxygenase reaction, measured using an electrochemical hydrogen peroxide sensor, and on protective hole hopping mechanisms, measured using stopped-flow ultraviolet–visible (UV–vis) spectrophotometry, differed from the AA9 LPMO. This shows that the impact of this Glu/Gln residue is dependent on additional structural or dynamic differences between the LPMOs. Despite the presence of several tryptophan residues in the protein core, the hole hopping studies revealed formation of only a tyrosyl feature with a lifespan distinct from similar features detected in other LPMOs, further highlighting the unique properties of AfAA11B.

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, using a single copper cofactor bound in a conserved histidine brace with a more variable second coordination sphere. Cellulose-active LPMOs in the fungal AA9 family and in a subset of bacterial AA10 enzymes contain a His-Gln-Tyr second sphere motif, whereas other cellulose-active AA10s have an Arg-Glu-Phe motif. To shine a light on the impact of this variation, we generated single, double, and triple mutations changing the His²¹⁶-Gln²¹⁹-Tyr²²¹ motif in cellulose- and chitin-oxidizing MaAA10B toward Arg-Glu-Phe. These mutations generally reduced enzyme performance due to rapid inactivation under turnover conditions, showing that catalytic fine-tuning of the histidine brace is complex and that the roles of these second sphere residues are strongly interconnected. Studies of copper reactivity showed remarkable effects, such as an increase in oxidase activity following the Q219E mutation and a strong dependence of this effect on the presence of Tyr at position 221. In reductant-driven reactions, differences in oxidase activity, which lead to different levels of in situ generated H₂O₂, correlated with differences in polysaccharide-degrading ability. The single Q219E mutant displayed a marked increase in activity on chitin in both reductant-driven reactions and reactions fueled by exogenously added H₂O₂. Thus, it seems that the evolution of substrate specificity in LPMOs involves both the extended substrate-binding surface and the second coordination sphere.

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein’s surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C-H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H₂O₂ splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.

Polysaccharide‐degrading mono‐copper lytic polysaccharide monooxygenases (LPMOs) are efficient peroxygenases that require electron donors (reductants) to remain in the active Cu(I) form and to generate the H₂O₂ co‐substrate from molecular oxygen. Here, we show how commonly used reductants affect LPMO catalysis in a pH‐dependent manner. Between pH 6.0 and 8.0, reactions with ascorbic acid show little pH dependency, whereas reactions with gallic acid become much faster at increased pH. These dependencies correlate with the reductant ionization state, which affects its ability to react with molecular oxygen and generate H₂O₂. The correlation does not apply to l‐cysteine because, as shown by stopped‐flow kinetics, increased H₂O₂ production at higher pH is counteracted by increased binding of l‐cysteine to the copper active site. The findings highlight the importance of the choice of reductant and pH in LPMO reactions.