The role of the active site tyrosine in the mechanism of lytic polysaccharide monooxygenase

McEvoy, Aina; Creutzberg, Joel; Singh, Raushan Kumar; Bjerrum, Morten J.; Hedegård, Erik D.

doi:10.1039/d0sc05262k

Cited by 21 publications

(44 citation statements)

References 56 publications

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“…In this study, we have shown that simply mimicking the transposition of the histidines or removing the carboxylate at the equatorial position is insufficient to recreate a redox-active copper site. This is in agreement with other studies of the importance of copper coordinating and second-sphere amino acid residues for LPMO activity [18,[40][41][42].…”

Section: Discussionsupporting

confidence: 93%

Copper binding and reactivity at the histidine brace motif: insights from mutational analysis of the Pseudomonas fluorescens copper chaperone CopC

et al. 2021

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The histidine brace (His-brace) is a copper-binding motif that is associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical and structural methods to characterize mutants of a Hisbrace-containing copper chaperone from Pseudomonas fluorescens (PfCopC). A total of 15 amino acid variants in primary and second-sphere residues were produced and characterized in terms of their copper binding and redox properties. PfCopC has a very high affinity for Cu(II) and also binds Cu(I). A high reorganization barrier likely prevents redox cycling and, thus, catalysis. In contrast, mutations in the conserved second-sphere Glu27 enable slow oxidation of ascorbate. The crystal structure of the variant E27A confirmed copper binding at the His-brace. Unexpectedly, Asp83 at the equatorial position was shown to be indispensable for Cu(II) binding in the His-brace of PfCopC. A PfCopC mutant that was designed to mimic the His-brace from lytic polysaccharide monooxygenase-like family X325 did not bind Cu(II), but was still able to bind Cu(I). These results highlight the importance of the proteinaceous environment around the copper His-brace for reactivity and, thus, the difference between enzyme and chaperone.

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Section: Discussionsupporting

confidence: 93%

Copper binding and reactivity at the histidine brace motif: insights from mutational analysis of the Pseudomonas fluorescens copper chaperone CopC

et al. 2021

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“…Several reports have suggested a higher affinity of at least some AA9 LPMOs for cellulosic substrates in their Cu + as opposed to their Cu 2+ form (Kracher et al, 2018;Bertini et al, 2018;Courtade et al, 2016;McEvoy et al, 2021). This has also been demonstrated recently for LsAA9_A (Brander, Tokin et al, 2021).…”

Section: Photoreduction Of Lsaa9_a Crystals Soaked With Oligosaccharidesmentioning

confidence: 69%

“…As well as being essential for further reactivity, there are several reports indicating increased polysaccharide affinity on reduction of the active-site metal, suggesting that polysaccharide binding precedes further steps in the mechanism (Kracher et al, 2018;Filandr et al, 2020;Brander, Tokin et al, 2021). Both O 2 and H 2 O 2 have been suggested as natural co-substrates (Bissaro et al, 2017; and the LPMO mechanism is being assessed continuously for the involvement of either/both (Bissaro et al, 2020;Hedega ˚rd & Ryde, 2017;Courtade et al, 2020;McEvoy et al, 2021;Brander et al, 2020). The dependence on H 2 O 2 for saccharide cleavage has been established recently specifically for one of the model LPMOs investigated here (Brander, Tokin et al, 2021), and is generally gaining increasing support.…”

mentioning

confidence: 99%

Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding

Tandrup¹,

Muderspach²,

Banerjee³

et al. 2022

Int Union Crystallogr J

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The recently discovered lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes capable of degrading polysaccharide substrates oxidatively. The generally accepted first step in the LPMO reaction is the reduction of the active-site metal ion from Cu2+ to Cu+. Here we have used a systematic diffraction data collection method to monitor structural changes in two AA9 LPMOs, one from Lentinus similis (LsAA9_A) and one from Thermoascus aurantiacus (TaAA9_A), as the active-site Cu is photoreduced in the X-ray beam. For LsAA9_A, the protein produced in two different recombinant systems was crystallized to probe the effect of post-translational modifications and different crystallization conditions on the active site and metal photoreduction. We can recommend that crystallographic studies of AA9 LPMOs wishing to address the Cu2+ form use a total X-ray dose below 3 × 104 Gy, while the Cu+ form can be attained using 1 × 106 Gy. In all cases, we observe the transition from a hexacoordinated Cu site with two solvent-facing ligands to a T-shaped geometry with no exogenous ligands, and a clear increase of the θ2 parameter and a decrease of the θ3 parameter by averages of 9.2° and 8.4°, respectively, but also a slight increase in θT. Thus, the θ2 and θ3 parameters are helpful diagnostics for the oxidation state of the metal in a His-brace protein. On binding of cello-oligosaccharides to LsAA9_A, regardless of the production source, the θT parameter increases, making the Cu site less planar, while the active-site Tyr—Cu distance decreases reproducibly for the Cu2+ form. Thus, the θT increase found on copper reduction may bring LsAA9_A closer to an oligosaccharide-bound state and contribute to the observed higher affinity of reduced LsAA9_A for cellulosic substrates.

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“…To address the observation of the two radical species, McEvoy et al performed QM/MM to model the feasibility of both deprotonated tyrosyl radical species. 64 The intermediate observed by Singh et al was proposed to be to a cis -[Tyr-CuOH] + formed by hydrogen transfer from the tyrosine hydroxy to an oxyl species in the equatorial plane. The intermediate observed by Paradisi et al was characterized as a trans -[Tyr-CuOH] + which can be formed through internal hydrogen transfer from a water molecule axially coordinated to the copper.…”

Section: Resultsmentioning

confidence: 99%

Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

et al. 2022

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The role of the active site tyrosine in the mechanism of lytic polysaccharide monooxygenase

Abstract: With QM/MM, we investigate the mechanism of tyrosine deprotonation in lytic polysaccharide monooxygenases. Our results support deprotonation and our calculated UV-vis spectra show that two isomers must be formed to match recent experiments.

Cited by 21 publications

References 56 publications

Copper binding and reactivity at the histidine brace motif: insights from mutational analysis of the Pseudomonas fluorescens copper chaperone CopC

Copper binding and reactivity at the histidine brace motif: insights from mutational analysis of the Pseudomonas fluorescens copper chaperone CopC

Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding

Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

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