Improved rate of substrate oxidation catalyzed by genetically-engineered myoglobin

Chand, Subhash; Ray, Sriparna; Wanigasekara, Eranda; Yadav, Poonam; Crawford, Joshua A.; Armstrong, Daniel W.; Rajeshwar, Krishnan; Pierce, Brad S.

doi:10.1016/j.abb.2017.12.014

Cited by 6 publications

(16 citation statements)

References 38 publications

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“…The WT Mb and H64DOPA Mb constructs were prepared following the previously described method [13][14][15]. In brief, the WT Mb expression construct and its H64TAG gene were cloned into an ampicillin resistance pBAD expression vector.…”

Section: Wild-type Mb and H64dopa Mb Mutant Constructs Preparationmentioning

confidence: 99%

“…Varian Cary 50 Bio UV-visible spectrophotometer was used to acquire UV-visible electronic absorption spectra of WT and H64DOPA Mb. WT Mb and mutant H64DOPA Mb protein concentrations were calculated from the absorption spectrum, using the molar extinction coefficient value of 170 mM −1 cm −1 at 409 nm and 411 nm, respectively [13][14][15]. The effect of hydrogen peroxide on ferric proteins UVvisible electronic absorption spectra was observed in the presence of 5 mM H 2 O 2 on 5 μM protein concentration at 20 °C (Figure 2).…”

Section: Spectroscopymentioning

confidence: 99%

“…A modulation frequency of 100 kHz was used for all EPR spectra. All data EPR data was collected under non-saturating conditions and simulated as described previsoulsy [13].…”

Section: Spectroscopymentioning

confidence: 99%

“…Myoglobin (Mb) is a stable heme-protein that can tolerate multiple mutations near its active site [10][11][12]. Previously we have genetically modified Mb by incorporating non-canonical amino acid at several positions to attain varied functions [13][14][15]. At the distal site of Mb, oxygen can directly bind to the heme-iron, while at the axial position, a histidine residue links the heme center to the protein moiety [16].…”

Section: Introductionmentioning

confidence: 99%

“…At the distal site of Mb, oxygen can directly bind to the heme-iron, while at the axial position, a histidine residue links the heme center to the protein moiety [16]. Various residues present in the vicinity of the active site are critical in determining the catalytic activity and electronic nature of the heme center [13,17,18]. A variety of metalloenzymes (peroxidases, catalases, cytochrome P450s, etc.)…”

Section: Introductionmentioning

confidence: 99%

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Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acid

Chand

Ray

Yadav

et al. 2021

Biochemical Journal

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To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compound I in the catalytic oxidation process by participating in an additional hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The reduction potential values of WT (E° = - 260 mV) and mutant Mb (E° = - 300 mV), w.r.t. Ag/AgCl reference electrode, in the presence of hydrogen peroxide, indicated an additional H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We observed that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, respectively, as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochemical studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.

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Section: Wild-type Mb and H64dopa Mb Mutant Constructs Preparationmentioning

confidence: 99%

Section: Spectroscopymentioning

confidence: 99%

“…A modulation frequency of 100 kHz was used for all EPR spectra. All data EPR data was collected under non-saturating conditions and simulated as described previsoulsy [13].…”

Section: Spectroscopymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acid

Chand

Ray

Yadav

et al. 2021

Biochemical Journal

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A novel thermophilic hemoprotein scaffold for rational design of biocatalysts

Aggrey-Fynn

Sürmeli

2018

J Biol Inorg Chem

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Hemoproteins are commonly found in nature, and involved in many important cellular processes such as oxygen transport, electron transfer, and catalysis. Rational design of hemoproteins can not only inspire novel biocatalysts but will also lead to a better understanding of structure-function relationships in native hemoproteins. Here, the heme nitric oxide/oxygen-binding protein from Caldanaerobacter subterraneus subsp. tengcongensis (TtH-NOX) is used as a novel scaffold for oxidation biocatalyst design. We show that signaling protein TtH-NOX can be reengineered to catalyze HO decomposition and oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) by HO. In addition, the role of the distal tyrosine (Tyr140) in catalysis is investigated. The mutation of Tyr140 to alanine hinders the catalysis of the oxidation reactions. On the other hand, the mutation of Tyr140 to histidine, which is commonly observed in peroxidases, leads to a significant increase of the catalytic activity. Taken together, these results show that, while the distal histidine plays an important role in hemoprotein reactions with HO, it is not always essential for oxidation activity. We show that TtH-NOX protein can be used as an alternative scaffold for the design of novel biocatalysts with desired reactivity or functionality. H-NOX proteins are homologous to the nitric oxide sensor soluble guanylate cyclase. Here, we show that the gas sensor protein TtH-NOX shows limited capacity for catalysis of redox reactions and it can be used as a novel scaffold in biocatalysis design.

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Noncanonical Amino Acids: Bringing New-to-Nature Functionalities to Biocatalysis

Brouwer,

Della-Felice,

Illies

et al. 2024

Chem. Rev.

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Biocatalysis has become an important component of modern organic chemistry, presenting an efficient and environmentally friendly approach to synthetic transformations. Advances in molecular biology, computational modeling, and protein engineering have unlocked the full potential of enzymes in various industrial applications. However, the inherent limitations of the natural building blocks have sparked a revolutionary shift. In vivo genetic incorporation of noncanonical amino acids exceeds the conventional 20 amino acids, opening new avenues for innovation. This review provides a comprehensive overview of applications of noncanonical amino acids in biocatalysis. We aim to examine the field from multiple perspectives, ranging from their impact on enzymatic reactions to the creation of novel active sites, and subsequent catalysis of new-to-nature reactions. Finally, we discuss the challenges, limitations, and promising opportunities within this dynamic research domain. CONTENTSAC 5.3. Photocatalytic Enzymes AG 5.3.1. ncAA as Bio-orthogonal Handle AG 5.3.2. ncAA as Metal-Binding Ligand or Photosensitizer AH 5.4. Discussion and Perspective on the Use of ncAAs in Artificial Enzyme Design AJ 6. Conclusions and Outlook

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Improved rate of substrate oxidation catalyzed by genetically-engineered myoglobin

Cited by 6 publications

References 38 publications

Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acid

Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acid

A novel thermophilic hemoprotein scaffold for rational design of biocatalysts

Noncanonical Amino Acids: Bringing New-to-Nature Functionalities to Biocatalysis

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