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1. Increasing Reduction Potentials of Type 1 Copper Center and Catalytic Efficiency of Small Laccase from Streptomyces coelicolor through Secondary Coordination Sphere Mutations

   https://doi.org/10.1002/anie.202314019  

   Wang, Jing‐Xiang ; Vilbert, Avery C. ; Cui, Chang ; Mirts, Evan N. ; Williams, Lucas H. ; Kim, Wantae ; Jessie Zhang, Y. ; Lu, Yi ( November 2023 , Angewandte Chemie International Edition)

   The key to type 1 copper (T1Cu) function lies in the fine tuning of the Cu^II/Ireduction potential (E°′_T1Cu) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tuneE°′_T1Cuin azurin over a wide range, these principles are yet to be generalized to other T1Cu‐containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) fromStreptomyces coelicolorto match the highE°′_T1Cuof fungal laccases. Using ultraviolet‐visible absorption and electron paramagnetic resonance spectroscopies, together with X‐ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and correspondingE°′_T1Cu. While minimal and smallE°′_T1Cuincreases are observed in Y229F‐ and S292F‐SLAC, the V290N mutant exhibits a majorE°′_T1Cuincrease. Moreover, the influence of these mutations onE°′_T1Cuis additive, culminating in a triple mutant Y229F/V290N/S292F‐SLAC with the highestE°′_T1Cuof 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (k_cat/K_M) relative to WT‐SLAC.

    

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2. Increasing Reduction Potentials of Type 1 Copper Center and Catalytic Efficiency of Small Laccase from Streptomyces coelicolor through Secondary Coordination Sphere Mutations

   https://doi.org/10.1002/ange.202314019  

   Wang, Jing‐Xiang ; Vilbert, Avery C. ; Cui, Chang ; Mirts, Evan N. ; Williams, Lucas H. ; Kim, Wantae ; Jessie Zhang, Y. ; Lu, Yi ( November 2023 , Angewandte Chemie)

   The key to type 1 copper (T1Cu) function lies in the fine tuning of the Cu^II/Ireduction potential (E°′_T1Cu) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tuneE°′_T1Cuin azurin over a wide range, these principles are yet to be generalized to other T1Cu‐containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) fromStreptomyces coelicolorto match the highE°′_T1Cuof fungal laccases. Using ultraviolet‐visible absorption and electron paramagnetic resonance spectroscopies, together with X‐ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and correspondingE°′_T1Cu. While minimal and smallE°′_T1Cuincreases are observed in Y229F‐ and S292F‐SLAC, the V290N mutant exhibits a majorE°′_T1Cuincrease. Moreover, the influence of these mutations onE°′_T1Cuis additive, culminating in a triple mutant Y229F/V290N/S292F‐SLAC with the highestE°′_T1Cuof 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (k_cat/K_M) relative to WT‐SLAC.

    

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3. A conformational equilibrium in the nitrogenase MoFe protein with an α-V70I amino acid substitution illuminates the mechanism of H 2 formation

   https://doi.org/10.1039/d2fd00153e  

   Lukoyanov, Dmitriy A. ; Yang, Zhi-Yong ; Shisler, Krista ; Peters, John W. ; Raugei, Simone ; Dean, Dennis R. ; Seefeldt, Lance C. ; Hoffman, Brian M. ( July 2023 , Faraday Discussions)

   Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe 7 S 9 MoC-homocitrate) as a critical N 2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E 4 (4H), which has accumulated 4[e − /H + ] as two bridging hydrides, Fe2–H–Fe6 and Fe3–H–Fe7, and protons bound to two sulfurs. E 4 (4H) is poised to bind/reduce N 2 as driven by mechanistically-coupled H 2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H 2 as the enzyme relaxes to state E 2 (2H), containing 2[e − /H + ] as a hydride and sulfur-bound proton; accumulation of E 4 (4H) in α-V70I is enhanced by HP suppression. EPR and 95 Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e − /H + ] to the E 0 state of the WT MoFe protein and to both α-V70I conformations generating E 2 (2H) that contains the Fe3–H–Fe7 bridging hydride; accumulation of another 2[e − /H + ] generates E 4 (4H) with Fe2–H–Fe6 as the second hydride. E 4 (4H) in WT enzyme and a minority α-V70I E 4 (4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2–H–Fe6 followed by slower HP of Fe3–H–Fe7, which leads to transient accumulation of E 2 (2H) containing Fe3–H–Fe7. In the dominant α-V70I E 4 (4H) conformation, HP of Fe2–H–Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3–H–Fe7 occurs first and the resulting E 2 (2H) contains Fe2–H–Fe6. It is this HP suppression in E 4 (4H) that enables α-V70I MoFe to accumulate E 4 (4H) in high occupancy. In addition, HP suppression in α-V70I E 4 (4H) kinetically unmasks hydride reductive-elimination without N 2 -binding, a process that is precluded in WT enzyme. 

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4. Conserved residues are critical for Haloferax volcanii archaeosortase catalytic activity: Implications for convergent evolution of the catalytic mechanisms of non‐homologous sortases from archaea and bacteria

   https://doi.org/10.1111/mmi.13935  

   Abdul Halim, Mohd Farid ; Rodriguez, Ronald ; Stoltzfus, Jonathan D. ; Duggin, Iain G. ; Pohlschroder, Mechthild ( March 2018 , Molecular Microbiology)

   Proper protein anchoring is key to the biogenesis of prokaryotic cell surfaces, dynamic, resilient structures that play crucial roles in various cell processes. A novel surface protein anchoring mechanism inHaloferax volcaniidepends upon the peptidase archaeosortase A (ArtA) processing C‐termini of substrates containing C‐terminal tripartite structures and anchoring mature substrates to the cell membrane via intercalation of lipid‐modified C‐terminal amino acid residues. While this membrane protein lacks clear homology to soluble sortase transpeptidases of Gram‐positive bacteria, which also process C‐termini of substrates whose C‐terminal tripartite structures resemble those of ArtA substrates, archaeosortases do contain conserved cysteine, arginine and arginine/histidine/asparagine residues, reminiscent of His‐Cys‐Arg residues of sortase catalytic sites. The study presented here shows that ArtA^WT‐GFP expressedin transcomplements ΔartAgrowth and motility phenotypes, while alanine substitution mutants, Cys¹⁷³(C173A), Arg²¹⁴(R214A) or Arg²⁵³(R253A), and the serine substitution mutant for Cys¹⁷³(C173S), fail to complement these phenotypes. Consistent with sortase active site replacement mutants, ArtA^C173A‐GFP, ArtA^C173S‐GFP and ArtA^R214A‐GFP cannot process substrates, while replacement of the third residue, ArtA^R253A‐GFP retains some processing activity. These findings support the view that similarities between certain aspects of the structures and functions of the sortases and archaeosortases are the result of convergent evolution.

    

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5. Are seven amino acid substitutions sufficient to explain the evolution of high l ‐DOPA 4,5‐dioxygenase activity leading to betalain pigmentation? Revisiting the gain‐of‐function mutants of Bean et al . (2018)

   https://doi.org/10.1111/nph.18981  

   Guerrero‐Rubio, M. Alejandra ; Walker‐Hale, Nathanael ; Guo, Rui ; Sheehan, Hester ; Timoneda, Alfonso ; Gandia‐Herrero, Fernando ; Brockington, Samuel F. ( September 2023 , New Phytologist)

   This work revisits a publication by Beanet al.(2018) that reports seven amino acid substitutions are essential for the evolution ofl‐DOPA 4,5‐dioxygenase (DODA) activity in Caryophyllales. In this study, we explore several concerns which led us to replicate the analyses of Beanet al.(2018).

   Our comparative analyses, with structural modelling, implicate numerous residues additional to those identified by Beanet al.(2018), with many of these additional residues occurring around the active site of BvDODAα1. We therefore replicated the analyses of Beanet al.(2018) to re‐observe the effect of their original seven residue substitutions in a BvDODAα2 background, that is the BvDODAα2‐mut3 variant.

   Multiplein vivoassays, in bothSaccharomyces cerevisiaeandNicotiana benthamiana, did not result in visible DODA activity in BvDODAα2‐mut3, with betalain production always 10‐fold below BvDODAα1.In vitroassays also revealed substantial differences in both catalytic activity and pH optima between BvDODAα1, BvDODAα2 and BvDODAα2‐mut3 proteins, explaining their differing performancein vivo.

   In summary, we were unable to replicate thein vivoanalyses of Beanet al.(2018), and our quantitativein vivoandin vitroanalyses suggest a minimal effect of these seven residues in altering catalytic activity of BvDODAα2. We conclude that the evolutionary pathway to high DODA activity is substantially more complex than implied by Beanet al.(2018).

    

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