Active site arginine controls the stereochemistry of hydride transfer in cyclohexanone monooxygenase

Fordwour, Osei Boakye; Wolthers, Kirsten R.

doi:10.1016/j.abb.2018.09.025

Cited by 6 publications

(4 citation statements)

References 39 publications

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“…In this scenario, the loss of arginine could have two counteracting effects: as quantum mechanics studies suggest that a nearby aspartate protonates the arginine and this stabilizes the negatively charged, deprotonated peroxyflavin, the arginine mutation could favor hydroperoxyflavin formation and thus the electrophilic mechanism. Contrarily, arginine loss decreases the overall reaction rate as the residue also promotes the reductive half-reaction and the rate of (hydro)peroxyflavin formation. , Interestingly, the substitution of a highly conserved aromatic residue with arginine was found in two independent studies that screened for variants with increased sulfoxidation activity. , In most BVMOs, this residue is a tryptophan that hydrogen bonds to the 3′ OH of the NADP ribose. Considering the enzyme’s tolerance of other aromatic residues at this position, this interaction is likely not influencing the electronics at the 2′ OH, which critically hydrogen bonds to the substrate carbonyl to activate it for nucleophilic attack (Scheme ).…”

Section: Promiscuous Catalytic Activitiesmentioning

confidence: 99%

“…Contrarily, arginine loss decreases the overall reaction rate as the residue also promotes the reductive half-reaction and the rate of (hydro)peroxyflavin formation. 76,142 Interestingly, the substitution of a highly conserved aromatic residue with arginine was found in two independent studies that screened for variants with increased sulfoxidation activity. 42,127 In most BVMOs, this residue is a tryptophan that hydrogen bonds to the 3′ OH of the NADP ribose.…”

Section: ■ Promiscuous Catalytic Activitiesmentioning

confidence: 99%

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Baeyer–Villiger Monooxygenases: Tunable Oxidative Biocatalysts

et al. 2019

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Pollution, accidents, and misinformation have earned the pharmaceutical and chemical industry a poor public reputation, despite their undisputable importance to society. Biotechnological advances hold the promise to enable a future of drastically reduced environmental impact and rigorously more efficient production routes at the same time. This is exemplified in the Baeyer–Villiger reaction, which offers a simple synthetic route to oxidize ketones to esters, but application is hampered by the requirement of hazardous and dangerous reagents. As an attractive alternative, flavin-containing Baeyer–Villiger monooxygenases (BVMOs) have been investigated for their potential as biocatalysts for a long time, and many variants have been characterized. After a general look at the state of biotechnology, we here summarize the literature on biochemical characterizations, mechanistic and structural investigations, as well as enzyme engineering efforts in BVMOs. With a focus on recent developments, we critically outline the advances toward tuning these enzymes suitable for industrial applications.

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Section: Promiscuous Catalytic Activitiesmentioning

confidence: 99%

Section: ■ Promiscuous Catalytic Activitiesmentioning

confidence: 99%

Baeyer–Villiger Monooxygenases: Tunable Oxidative Biocatalysts

et al. 2019

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“…This structural geometry and the Arg/Asp pair were conserved in Baeyer-Villiger monooxygenases, including steroid monooxygenase (STMO) and phenylacetone monooxygenase (PAMO) [58,59]. The elimination of interaction between the Arg/Asp pair in the mutant of CHMO could prolong the lifetime of the C4a-OO − intermediate, and this was attributed to the substrateinduced conformation resulting in slowing-down diffusion of a substrate into the active site [60]. Based on this previous information, no clear evidence for the influence of assisting residues in the protonation status of C4a-OO(H) stabilization was identified.…”

Section: Discussionmentioning

confidence: 99%

Formation and stabilization of C4a‐hydroperoxy‐FAD by the Arg/Asn pair in HadA monooxygenase

Pimviriyakul

Chaiyen

2022

The FEBS Journal

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HadA monooxygenase catalyses the detoxification of halogenated phenols and nitrophenols via dehalogenation and denitration respectively. C4a‐hydroperoxy‐FAD is a key reactive intermediate wherein its formation, protonation and stabilization reflect enzyme efficiency. Herein, transient kinetics, site‐directed mutagenesis and pH‐dependent behaviours of HadA reaction were employed to identify key features stabilizing C4a‐adducts in HadA. The formation of C4a‐hydroperoxy‐FAD is pH independent, whereas its decay and protonation of distal oxygen are associated with pKa values of 8.5 and 8.4 respectively. These values are correlated with product formation within a pH range of 7.6–9.1, indicating the importance of adduct stabilization to enzymatic efficiency. We identified Arg101 as a key residue for reduced FAD (FADH−) binding and C4a‐hydroperoxy‐FAD formation due to the loss of these abilities as well as enzyme activity in HadAR101A and HadAR101Q. Mutations of the neighbouring Asn447 do not affect the rate of C4a‐hydroperoxy‐FAD formation; however, they impair FADH− binding. The disruption of Arg101/Asn447 hydrogen bond networking in HadAN447A increases the pKa value of C4a‐hydroperoxy‐FAD decay to 9.5; however, this pKa was not altered in HadAN447D (pKa of 8.5). Thus, Arg101/Asn447 pair should provide important interactions for FADH− binding and maintain the pKa associated with H2O2 elimination from C4a‐hydroperoxy‐FAD in HadA. In the presence of substrate, the formation of C4a‐hydroxy‐FAD at the hydroxylation step is pH insensitive, and it dehydrates to form the oxidized FAD with pKa of 7.9. This structural feature might help elucidate how the reactive intermediate was stabilized in other flavin‐dependent monooxygenases.

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“…For these reasons, NMR spectroscopy is highly sought after in drug development [ 37 , 38 , 39 , 40 , 41 ], for both molecule identification [ 11 , 13 , 14 , 18 , 42 , 43 , 44 , 45 , 46 ] and structural elucidation [ 15 , 16 , 17 , 45 , 47 , 48 , 49 , 50 , 51 ]. NMR has been successfully applied in stereochemistry [ 52 , 53 , 54 , 55 , 56 ] and isomer determination [ 57 , 58 , 59 , 60 , 61 ], in drug-protein interactions studies [ 62 , 63 , 64 ], and in the evaluation of drug toxicity [ 65 , 66 , 67 , 68 ].…”

Section: Introductionmentioning

confidence: 99%

NMR as a “Gold Standard” Method in Drug Design and Discovery

et al. 2020

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Studying disease models at the molecular level is vital for drug development in order to improve treatment and prevent a wide range of human pathologies. Microbial infections are still a major challenge because pathogens rapidly and continually evolve developing drug resistance. Cancer cells also change genetically, and current therapeutic techniques may be (or may become) ineffective in many cases. The pathology of many neurological diseases remains an enigma, and the exact etiology and underlying mechanisms are still largely unknown. Viral infections spread and develop much more quickly than does the corresponding research needed to prevent and combat these infections; the present and most relevant outbreak of SARS-CoV-2, which originated in Wuhan, China, illustrates the critical and immediate need to improve drug design and development techniques. Modern day drug discovery is a time-consuming, expensive process. Each new drug takes in excess of 10 years to develop and costs on average more than a billion US dollars. This demonstrates the need of a complete redesign or novel strategies. Nuclear Magnetic Resonance (NMR) has played a critical role in drug discovery ever since its introduction several decades ago. In just three decades, NMR has become a “gold standard” platform technology in medical and pharmacology studies. In this review, we present the major applications of NMR spectroscopy in medical drug discovery and development. The basic concepts, theories, and applications of the most commonly used NMR techniques are presented. We also summarize the advantages and limitations of the primary NMR methods in drug development.

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Active site arginine controls the stereochemistry of hydride transfer in cyclohexanone monooxygenase

Cited by 6 publications

References 39 publications

Baeyer–Villiger Monooxygenases: Tunable Oxidative Biocatalysts

Baeyer–Villiger Monooxygenases: Tunable Oxidative Biocatalysts

Formation and stabilization of C4a‐hydroperoxy‐FAD by the Arg/Asn pair in HadA monooxygenase

NMR as a “Gold Standard” Method in Drug Design and Discovery

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