The role of conformational flexibility in Baeyer-Villiger monooxygenase catalysis and structure

Yachnin, Brahm J.; Lau, Peter C. K.; Berghuis, Albert M.

doi:10.1016/j.bbapap.2016.08.015

Cited by 22 publications

(21 citation statements)

References 28 publications

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“…These selected models were overlapped with their respective template structures for structural comparison (Figure 2A). The structural models of the putative BVMOs showed overall folds similar to those of template structures, with differences in some of the loops that connect α-helixes or β-strands, especially in the so-called “Control Loop” [27] (Figure 2A). This loop influences the active site environment and plays a critical role in enzyme structure and catalysis, mediating NADPH (Nicotinamide Adenine Dinucleotide Phosphate, hydride reduced form) binding and substrate selection [27].…”

Section: Resultsmentioning

confidence: 99%

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

et al. 2017

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The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer–Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.

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Section: Resultsmentioning

confidence: 99%

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

et al. 2017

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“…Surprisingly, an altered selectivity was observed with the four substrates tested [9]. This, together with small-angle X-ray scattering (SAXS) and targeted mutation studies, suggests that the control loop has a more complex role, affecting not only overall structure, but also the active site environment [10].…”

Section: Introductionmentioning

confidence: 92%

“…Mechanistically, enzyme-bound FAD is reduced by NAD(P)H, and after molecular oxygen is activated through the formation of a peroxyflavin, which then nucleophilically adds to the carbonyl group of the substrate. The reaction mechanism and structure of type I BVMOs have been extensively studied, with cyclohexanone monooxygenase (CHMO) [7][8][9][10] and phenylacetone monooxygenase (PAMO) [11][12][13] typically serving as the prototypes. Co-crystallization studies have revealed BVMOs to be highly flexible enzymes, able to undergo large domain movements during catalysis.…”

Section: Introductionmentioning

confidence: 99%

“…Post BV oxidation, the loop relaxes, allowing the product to diffuse out of the active site. As NADP(H) remains bound during the entire catalytic cycle, the conformation of the 'control loop' is crucial in understanding its role during catalysis [10,14]. Central to the 'control loop' is a conserved tryptophan (W492 in CHMO) which interacts with NADP(H).…”

Section: Introductionmentioning

confidence: 99%

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Natural Variation in the ‘Control Loop’ of BVMOAFL210 and Its Influence on Regioselectivity and Sulfoxidation

et al. 2020

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Baeyer-Villiger monooxygenases (BVMOs) are flavin-dependent enzymes that primarily convert ketones to esters, but can also catalyze heteroatom oxidation. Several structural studies have highlighted the importance of the 'control loop' in BVMOs, which adopts different conformations during catalysis. Central to the 'control loop' is a conserved tryptophan that has been implicated in NADP(H) binding. BVMO AFL210 from Aspergillus flavus, however, contains a threonine in the equivalent position. Here, we report the structure of BVMO AFL210 in complex with NADP + in both the 'open' and 'closed' conformations. In neither conformation does Thr513 contact the NADP + . Although mutagenesis of Thr513 did not significantly alter the substrate scope, changes in peroxyflavin stability and reaction rates were observed. Mutation of this position also brought about changes in the regioand enantioselectivity of the enzyme. Moreover, lower rates of overoxidation during sulfoxidation of thioanisole were also observed.

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“…The control loop is observed to be ordered in some CHMO crystal structures but disordered in others 21,23,36 , suggesting that its flexibility varies depending on the enzyme's position in catalytic cycle. Specifically, it is hypothesized that the control loop must become rigid to hold the NADPH cofactor and cyclohexanone during the hydride transfer stages 21 . We analyzed the flexibility of the control loop through α -carbon root mean square fluctuation (RMSF), and plot the difference between CHMO DTNP (bound with NADH) and CHMO WT (bound with NADH or NADPH, respectively) ( Figure 5).…”

Section: The Effect Of Cofactor Specificity-switching Mutations On Prmentioning

confidence: 99%

Pairing two growth-based, high-throughput selections to fine tune conformational dynamics in oxygenase engineering

Maxel

Zhang

King

et al. 2020

Preprint

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Cyclohexanone monooxygenases (CHMO) consume molecular oxygen and NADPH to catalyze the valuable oxidation of cyclic ketones. However, CHMO usage is restricted by poor thermostability and stringent specificity for NADPH. Efforts to engineer CHMO have been limited by the sensitivity of the enzyme to perturbations in conformational dynamics and long-range interactions that cannot be predicted. We demonstrate a pair of aerobic, high-throughput growth selection platforms in Escherichia coli for oxygenase evolution, based on NADPH or NADH redox balance. We utilize the NADPH-dependent selection in the directed evolution of thermostable CHMO and discover the variant CHMO GV (A245G-A288V) with a 2.7-fold improvement in residual activity compared to the wild type after 40 °C incubation. Addition of a previously reported mutation resulted in A245G-A288V-T415C which has further improved thermostability at 45 °C. We apply the NADH-dependent selection to alter the cofactor specificity of CHMO to accept NADH, a less expensive cofactor than NADPH. We identified the variant CHMO DTNP (S208D-K326T-K349N-L143P) with a 21-fold cofactor specificity switch from NADPH to NADH compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and backbone torsions, and CHMO DTNP activity is driven by cooperative fine-tuning of cofactor contacts. Our introduced tools for oxygenase evolution enable the rapid engineering of properties critical to industrial scalability. KEY WORDScyclohexanone monooxygenase, Acinetobacter sp., Baeyer-Villiger monooxygenase, directed evolution, thermostability, NAD(P)H cofactor specificity, redox balance, high-throughput selection Recently, we described the construction and application of an aerobic growth-based, highthroughput selection platform for engineering NADPH-dependent oxygenases 10 . We demonstrated that this selection platform enabled rapid remodeling of the Pseudomonas aeruginosa 4-hydroxybenzoate hydroxylase (PobA) active site to efficiently accept a non-native substrate 3,4-dihydroxybenzoic acid (3,. The selected variants appear to recognize the new substrate with synergistic hydrogen bond networks, which are difficult to discover without high-throughput searching of protein sequence space. However, the key limitation of this method is that it is not compatible with engineering NADH-dependent oxygenases. While NADPH-dependent oxygenases such as most P450s naturally function in anabolism, the NADH-dependent oxygenases function in catabolism and enable the conversion of various recalcitrant substrates such as xylene 11 and plastics 12 . Here, we report an aerobic growth-based selection for NADH activity, which complements the NADPH-dependent selection platform. Together, these two selection systems may support the full range of engineering capabilities to develop catalytically important oxygenases. SUPPORTING INFORMATIONExperimental and computational methods, plasmids and strains used in this study (Table S1), detailed kinetic analysis of CHMO variants...

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The role of conformational flexibility in Baeyer-Villiger monooxygenase catalysis and structure

Abstract: A better understanding of the mechanistic role of the Control Loop may ultimately be helpful in designing mutants with altered specificity and improved catalytic efficiency.

Cited by 22 publications

References 28 publications

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

Natural Variation in the ‘Control Loop’ of BVMOAFL210 and Its Influence on Regioselectivity and Sulfoxidation

Pairing two growth-based, high-throughput selections to fine tune conformational dynamics in oxygenase engineering

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