Advances in Engineered Hemoproteins that Promote Biocatalysis

Stone, Kari L.; Ahmed, Syeda M.

doi:10.3390/inorganics4020012

Cited by 5 publications

(2 citation statements)

References 82 publications

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“…Artificial metalloenzymes based on hemoproteins have gained considerable attention in the last few years as they show a promising new generation biocatalyst possessing specific catalytic activity with high efficiency [1][2][3]. One of the reasons behind choosing the hemoproteins for such biocatalysts is that they demonstrate a wide range of diverse but distinctive functions in the biological system [4][5][6][7].…”

Section: Introductionmentioning

confidence: 99%

An engineered thermally tolerant apo-cytochrome scaffold for metal-less incorporation of heme derivative

Ben Aoun,

Ibrahim

2023

PLoS ONE

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Cytochrome c552 from Thermus thermophilus is one of the hot topics for creating smart biomaterials as it possesses remarkable stability, is tolerant to multiple mutations and has therefore been recently reported for a number of functionalizations upon substitution of the original prosthetic group with an artificial prosthetic group. However, all of the substitutions were driven by the coordination through the axial ligands followed by complete reconstitution with a metal–porphyrin complex. This limits the scope of the cytochrome c for incorporating a metal-less non-natural heme species that could improve the versatility of cytochrome c for a new generation of engineered cytochrome proteins for further enhancement in their functionalities such as biocatalysts. In this connection, a new variant of Cytochrome c (rC552 C14A) from Thermus thermophilus was reported, where an easy approach to remove the original prosthetic group was achieved, followed by the incorporation of a number of metal-PPIX derivatives that ultimately led to the formation of artificial c-type cytochromes through covalent bonding. The apo-cytochrome was found to be thermally tolerant and to possess a distinctive overall structure as that of the wild type, as was evident from the corresponding CD spectra, which ultimately encouraged reconstitution with a metal-less protoporphyrin derivative for better understanding the role of axial ligands in the reconstitution process. Successful reconstitution was achieved, resulting in a new type of Cytochrome b-type artificial protein without the metal in its active site, indicating the non-involvement of the axial ligand. In order to prove the non-involvement of the axial ligand, a subsequent double mutant (C14A/M69A) was constructed, replacing the methionine at 69 position with non-coordinating alanine residue. Accordingly, the apo-C14A/M69A was prepared and found to be extremely stable as the earlier mutants and the WT showed no signs of denaturation, even at the elevated temperature of 98°C. Subsequently, heme b was successfully incorporated into the apo-C14A/M69A, which demonstrated itself as a highly thermally tolerant protein scaffold for incorporating a metal-less artificial prosthetic group in the absence of the axial ligand. Further improvement in the reconstitution process is achieved by replacing the methionine at 69 position with phenyl alanine (C14A/M69F mutant), resulting in further stabilization of heme species, possibly through non-covalent π–interactions, as corroborated by molecular docking.

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Section: Introductionmentioning

confidence: 99%

An engineered thermally tolerant apo-cytochrome scaffold for metal-less incorporation of heme derivative

Ben Aoun,

Ibrahim

2023

PLoS ONE

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“…Direct involvement of the heme in protein and enzyme function has prompted substitution of the iron protoporphyrin (FePPIX) 2 with protoporphyrins containing other metals. For decades, this approach has been used to obtain insight into enzyme mechanism, to generate novel enzyme activities (1)(2)(3)(4), and to facilitate structural studies (5). Many native heme-containing proteins can be produced in Escherichia coli either by relying on bacterial heme biosynthesis, by adding heme precursors during recombinant expression, or by expressing the apoprotein and reconstituting with PPIX during purification.…”

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confidence: 99%

Endogenous insertion of non-native metalloporphyrins into human membrane cytochrome P450 enzymes

Yadav

Scott

2018

Journal of Biological Chemistry

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Human cytochrome P450 enzymes are membrane-bound heme-containing monooxygenases. As is the case for many heme-containing enzymes, substitution of the metal in the center of the heme can be useful for mechanistic and structural studies of P450 enzymes. For many heme proteins, the iron protoporphyrin prosthetic group can be extracted and replaced with protoporphyrin containing another metal, but human membrane P450 enzymes are not stable enough for this approach. The method reported herein was developed to endogenously produce human membrane P450 proteins with a nonnative metal in the heme. This approach involved coexpression of the P450 of interest, a heme uptake system, and a chaperone in growing in iron-depleted minimal medium supplemented with the desired trans-metallated protoporphyrin. Using the steroidogenic P450 enzymes CYP17A1 and CYP21A2 and the drug-metabolizing CYP3A4, we demonstrate that this approach can be used with several human P450 enzymes and several different metals, resulting in fully folded proteins appropriate for mechanistic, functional, and structural studies including solution NMR.

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Catalytic Promiscuity of Horseradish Peroxidase: Aerobic Oxidative Regeneration of Oxidized Nicotinamide Cofactors

et al. 2023

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NAD(P)+ regeneration is of great importance for dehydrogenase (DH)‐catalyzed oxidations. In this work, we report horseradish peroxidase (HRP)‐catalyzed aerobic oxidation of the reduced nicotinamide cofactors, including natural and non‐natural ones, into the oxidized form at neutral and alkaline pH enabled by external amino acids (AAs). HRP showed promiscuous yet extremely low oxidase activity toward NADH at pH 7 in the absence of both AAs and mediators, as evidenced by the observation of compound III. The mechanistic study revealed that AAs as decoy molecules (likely act as acid‐base catalysts) and mediators significantly promoted the formation of compound III as well as its decay back to the ground state. The applicability of the aerobic cofactor regeneration method was demonstrated by the HRP total turnover number up to 88 000 as well as high yields (91–99 %) in bi‐enzymatic synthesis of carboxylic acids. The present work offers an alternative method to recycle NAD(P)+. Also, it may open up possibilities for HRP‐catalyzed aerobic oxidations.

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Advances in Engineered Hemoproteins that Promote Biocatalysis

Cited by 5 publications

References 82 publications

An engineered thermally tolerant apo-cytochrome scaffold for metal-less incorporation of heme derivative

An engineered thermally tolerant apo-cytochrome scaffold for metal-less incorporation of heme derivative

Endogenous insertion of non-native metalloporphyrins into human membrane cytochrome P450 enzymes

Catalytic Promiscuity of Horseradish Peroxidase: Aerobic Oxidative Regeneration of Oxidized Nicotinamide Cofactors

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