Chromogenic nitrophenolate-based substrates for light-driven hybrid P450 BM3 enzyme assay

Lam, Quan; Cortez, Alejandro; Nguyen, Thanh Truc; Kato, Mallory; Cheruzel, Lionel

doi:10.1016/j.jinorgbio.2015.12.005

Cited by 20 publications

(33 citation statements)

References 34 publications

(63 reference statements)

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“…The dF393A- 1 mutant showed minimal photocatalytic activity, while the dF393W- 1 shows a k cat of 21 eq/min and TTN of 192, a 20% decrease in activity compared to the values determined for the sL407C- 1 . [15] The k cat for the dQ403W- 1 is 42 eq/min and the TTN values are 60% higher (420) than those observed for the sL407C- 1 .…”

Section: Resultsmentioning

confidence: 98%

“…We used a colorimetric assay compatible with the light-driven conditions[15] to determine the Michaelis-Menten parameters and total turnover numbers (TTN) at the end of the reaction for the four hybrid enzymes in the hydroxylation of the long chain fatty acid analog, 16-pNCA, upon visible light-irradiation. The data are summarized in Table 2 and Figure S4.…”

Section: Resultsmentioning

confidence: 99%

“…The main challenges still reside in the timely delivery of the electrons to achieve high activity and limit the unproductive pathways [37, 38]. The light-driven approach used in the hybrid P450 enzymes[5, 6, 8, 9, 15] is taking advantages of the photochemical properties of Ru(II)-diimine photosensitizers and the extensive electron transfer studies in metalloproteins. [7]…”

Section: Discussionmentioning

confidence: 99%

“…[15] The following primers were used to create the desired mutations: F393A 5′-GCGTTTAAACCG GCT GGAAACGGTCAG-3′; F393W 5′-GCGTTTAAACCG TGG GGAAACGGTC AG-3′; Q403W 5′-CGTGTATCGGT TGG CAGTTCGCTTGC-3′. The underlined regions indicate the section of the oligonucleotides modified from the sL407C sequence to produce the codon change required for the desired amino acid substitutions.…”

Section: Methodsmentioning

confidence: 99%

“…A colorimetric assay compatible with the light-driven process was used to assess the photocatalytic activity of the double mutants relative to the sL407C- 1 . [15] Photocatalytic activity is negligible in the dF393A- 1 enzyme while a 60% increase in activity with total turnover numbers of 420 and 90% product conversion is observed in the dQ403W- 1 enzyme.…”

Section: Introductionmentioning

confidence: 99%

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Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies

Spradlin

Lee

Mahadevan

et al. 2016

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Background In order to perform selective C-H functionalization upon visible light irradiation, Ru(II)-diimine functionalized P450 heme enzymes have been developed. The sL407C-1 enzyme containing the Ru(bpy)2PhenA (bpy = 2,2′-bipyridine and PhenA = 5-acetamido-1,10-phenanthroline) photosensitizer (1) covalently attached to the non-native single cysteine L407C of the P450BM3 heme domain mutant, displays high photocatalytic activity in the selective C-H bond hydroxylation of several substrates. Methods A combination of X-ray crystallography, site-directed mutagenesis, transient absorption measurements and enzymatic assays was used to gain insights into its photocatalytic activity and electron transfer pathway. Results The crystal structure of the sL407C-1 enzyme was solved in the open and closed conformations revealing a through-space electron transfer pathway involving highly conserved, F393 and Q403, residues. Several mutations of these residues (F393A, F393W or Q403W) were introduced to probe their roles in the overall reaction. Transient absorption measurements confirm rapid electron transfer as heme reduction is observed in all four hybrid enzymes. Compared to the parent sL407C-1, photocatalytic activity was negligible in the dF393A-1 enzyme while 60% increase in activity with total turnover numbers of 420 and 90% product conversion was observed with the dQ403W-1 mutant. Conclusions In the sL407C-1 enzyme, the photosensitizer is ideally located to rapidly deliver electrons, using the naturally occurring electron transfer pathway, to the heme center in order to activate molecular dioxygen and sustain photocatalytic activity. General Significance The results shed light on the design of efficient light-driven biocatalysts and the approach can be generalized to other members of the P450 superfamily.

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies

Spradlin

Lee

Mahadevan

et al. 2016

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Light‐driven catalysis with engineered enzymes and biomimetic systems

Edwards

Bren

2020

Biotech and App Biochem

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Efforts to drive catalytic reactions with light, inspired by natural processes like photosynthesis, have a long history and have seen significant recent growth. Successfully engineering systems using biomolecular and bioinspired catalysts to carry out light‐driven chemical reactions capitalizes on advantages offered from the fields of biocatalysis and photocatalysis. In particular, driving reactions under mild conditions and in water, in which enzymes are operative, using sunlight as a renewable energy source yield environmentally friendly systems. Furthermore, using enzymes and bioinspired systems can take advantage of the high efficiency and specificity of biocatalysts. There are many challenges to overcome to fully capitalize on the potential of light‐driven biocatalysis. In this mini‐review, we discuss examples of enzymes and engineered biomolecular catalysts that are activated via electron transfer from a photosensitizer in a photocatalytic system. We place an emphasis on selected forefront chemical reactions of high interest, including CH oxidation, proton reduction, water oxidation, CO2 reduction, and N2 reduction.

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Autodisplay of glucose‐6‐phosphate dehydrogenase for redox cofactor regeneration at the cell surface

Schüürmann

Quehl

Lindhorst

et al. 2017

Biotech & Bioengineering

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Inherent cofactor regeneration is a pivotal feature of whole cell biocatalysis. For specific biotechnological applications, surface display of enzymes is emerging as a tool to circumvent mass transfer limitations or enzyme stability problems. Even complex reactions can be accomplished applying displayed enzymes. Yet, industrial utilization of the technique is still impeded by lacking cofactor regeneration at the cell surface. Here, we report on the surface display of a glucose-6-phoshate dehydrogenase (G6PDH) via Autodisplay to address this limitation and regenerate NADPH directly at the cell surface. The obtained whole cell biocatalyst demonstrated similar kinetic parameters compared to the purified enzyme, more precisely K values of 0.2 mM for NADP and calculated total turnover numbers of 10 . However, the K for the substrate G6P increased by a factor of 7 to yield 1.5 mM. The whole cell biocatalyst was cheaper to produce, easy to separate from the reaction mixture and reusable in consecutive reaction cycles. Furthermore, lyophilization allowed storage at room temperature. The whole cell biocatalyst displaying G6PDH was applicable for NADPH regeneration in combination with soluble as well as surface displayed enzymes and model reactions in combination with bacterial CYP102A1 and human CYP1A2 were realized. Biotechnol. Bioeng. 2017;114: 1658-1669. © 2017 Wiley Periodicals, Inc.

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Chromogenic nitrophenolate-based substrates for light-driven hybrid P450 BM3 enzyme assay

Cited by 20 publications

References 34 publications

Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies

Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies

Light‐driven catalysis with engineered enzymes and biomimetic systems

Autodisplay of glucose‐6‐phosphate dehydrogenase for redox cofactor regeneration at the cell surface

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