Publications by authors named "Marco W Fraaije"

168 Publications

Introducing an Artificial Deazaflavin Cofactor in and .

ACS Synth Biol 2022 Jan 19. Epub 2022 Jan 19.

Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands.

Deazaflavin-dependent whole-cell conversions in well-studied and industrially relevant microorganisms such as and have high potential for the biocatalytic production of valuable compounds. The artificial deazaflavin FOP (FO-5'-phosphate) can functionally substitute the natural deazaflavin F and can be synthesized in fewer steps, offering a solution to the limited availability of the latter due to its complex (bio)synthesis. Herein we set out to produce FOP in vivo as a scalable FOP production method and as a means for FOP-mediated whole-cell conversions. Heterologous expression of the riboflavin kinase from enabled in vivo phosphorylation of FO, which was supplied by either organic synthesis ex vivo, or by a coexpressed FO synthase in vivo, producing FOP in as well as in . Through combined approaches of enzyme engineering as well as optimization of expression systems and growth media, we further improved the in vivo FOP production in both organisms. The improved FOP production yield in is comparable to the F yield of native F-producing organisms such as , but the former can be achieved in a significantly shorter time frame. Our expression system has an estimated production rate of 0.078 μmol L h and results in an intracellular FOP concentration of about 40 μM, which is high enough to support catalysis. In fact, we demonstrate the successful FOP-mediated whole-cell conversion of ketoisophorone using cells. In , in vivo FOP production by RFK using supplied FO was improved through media optimization and enzyme engineering. Through structure-guided enzyme engineering, a RFK variant with 7-fold increased catalytic efficiency compared to the wild type was discovered. By using this variant in optimized media conditions, FOP production yield in was 20-fold increased compared to the very low initial yield of 0.24 ± 0.04 nmol per g dry biomass. The results show that bacterial and eukaryotic hosts can be engineered to produce the functional deazaflavin cofactor mimic FOP.
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http://dx.doi.org/10.1021/acssynbio.1c00552DOI Listing
January 2022

Discovery of Two Novel Oxidases Using a High-Throughput Activity Screen.

Chembiochem 2022 Jan 18;23(2):e202100510. Epub 2021 Nov 18.

Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96, Gothenburg, Sweden.

Discovery of novel enzymes is a challenging task, yet a crucial one, due to their increasing relevance as chemical catalysts and biotechnological tools. In our work we present a high-throughput screening approach to discovering novel activities. A screen of 96 putative oxidases with 23 substrates led to the discovery of two new enzymes. The first enzyme, N-acetyl-D-hexosamine oxidase (EC 1.1.3.29) from Ralstonia solanacearum, is a vanillyl alcohol oxidase-like flavoprotein displaying the highest activity with N-acetylglucosamine and N-acetylgalactosamine. Before our discovery of the enzyme, its activity was an orphan one - experimentally characterized but lacking the link to amino acid sequence. The second enzyme, from an uncultured marine euryarchaeota, is a long-chain alcohol oxidase (LCAO, EC 1.1.3.20) active with a range of fatty alcohols, with 1-dodecanol being the preferred substrate. The enzyme displays no sequence similarity to previously characterised LCAOs, and thus is a completely novel representative of a protein with such activity.
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http://dx.doi.org/10.1002/cbic.202100510DOI Listing
January 2022

A Tailor-Made Deazaflavin-Mediated Recycling System for Artificial Nicotinamide Cofactor Biomimetics.

ACS Catal 2021 Sep 2;11(18):11561-11569. Epub 2021 Sep 2.

Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands.

Nicotinamide adenine dinucleotide (NAD) and its 2'-phosphorylated form NADP are crucial cofactors for a large array of biocatalytically important redox enzymes. Their high cost and relatively poor stability, however, make them less attractive electron mediators for industrial processes. Nicotinamide cofactor biomimetics (NCBs) are easily synthesized, are inexpensive, and are also generally more stable than their natural counterparts. A bottleneck for the application of these artificial hydride carriers is the lack of efficient cofactor recycling methods. Therefore, we engineered the thermostable F:NADPH oxidoreductase from (FNO), by structure-inspired site-directed mutagenesis, to accommodate the unnatural N1 substituents of eight NCBs. The extraordinarily low redox potential of the natural cofactor FH was then exploited to reduce these NCBs. Wild-type enzyme had detectable activity toward all selected NCBs, with values in the millimolar range and values ranging from 0.09 to 1.4 min. Saturation mutagenesis at positions Gly-29 and Pro-89 resulted in mutants with up to 139 times higher catalytic efficiencies. Mutant G29W showed a value of 4.2 s toward 1-benzyl-3-acetylpyridine (BAP), which is similar to the value for the natural substrate NADP. The best FNO variants for a specific NCB were then used for the recycling of catalytic amounts of these nicotinamides in conversion experiments with the thermostable ene-reductase from (OYE). We were able to fully convert 10 mM ketoisophorone with BAP within 16 h, using F or its artificial biomimetic FOP (FO-2'-phosphate) as an efficient electron mediator and glucose-6-phosphate as an electron donor. The generated toolbox of thermostable and NCB-dependent FNO variants offers powerful cofactor regeneration biocatalysts for the reduction of several artificial nicotinamide biomimetics at both ambient and high temperatures. In fact, to our knowledge, this enzymatic method seems to be the best-performing NCB-recycling system for BNAH and BAPH thus far.
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http://dx.doi.org/10.1021/acscatal.1c03033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8453485PMC
September 2021

Discovery, Biocatalytic Exploration and Structural Analysis of a 4-Ethylphenol Oxidase from Gulosibacter chungangensis.

Chembiochem 2021 Nov 30;22(22):3225-3233. Epub 2021 Sep 30.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Via Ferrata 9, 27100, Pavia, Italy.

The vanillyl-alcohol oxidase (VAO) family is a rich source of biocatalysts for the oxidative bioconversion of phenolic compounds. Through genome mining and sequence comparisons, we found that several family members lack a generally conserved catalytic aspartate. This finding led us to study a VAO-homolog featuring a glutamate residue in place of the common aspartate. This 4-ethylphenol oxidase from Gulosibacter chungangensis (Gc4EO) shares 42 % sequence identity with VAO from Penicillium simplicissimum, contains the same 8α-N -histidyl-bound FAD and uses oxygen as electron acceptor. However, Gc4EO features a distinct substrate scope and product specificity as it is primarily effective in the dehydrogenation of para-substituted phenols with little generation of hydroxylated products. The three-dimensional structure shows that the characteristic glutamate side chain creates a closely packed environment that may limit water accessibility and thereby protect from hydroxylation. With its high thermal stability, well defined structural properties and high expression yields, Gc4EO may become a catalyst of choice for the specific dehydrogenation of phenolic compounds bearing small substituents.
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http://dx.doi.org/10.1002/cbic.202100457DOI Listing
November 2021

SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices.

Int J Mol Sci 2021 Jul 27;22(15). Epub 2021 Jul 27.

Instituto de Tecnologia Química e Biológica António Xavier, Universidade NOVA de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.

Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of HO. To this end, these enzymes have to preserve native, solution properties upon immobilisation on the electrode surface. In this work, DyPs from (CboDyP), (ScoDyP) and (TfuDyP) are immobilised on biocompatible silver electrodes functionalized with alkanethiols. Their structural, redox and catalytic properties upon immobilisation are evaluated by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and cyclic voltammetry. Among the studied electrode/DyP constructs, only CboDyP shows preserved native structure upon attachment to the electrode. However, a comparison of the redox potentials of the enzyme in solution and immobilised states reveals a large discrepancy, and the enzyme shows no electrocatalytic activity in the presence of HO. While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the immobilised state. The capacity of SERR spectroelectrochemistry for fast screening of the performance of immobilised heme enzymes places it in the front-line of experimental approaches that can advance the search for promising DyP candidates.
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http://dx.doi.org/10.3390/ijms22157998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8348443PMC
July 2021

Flavin-tag: A Facile Method for Site-Specific Labeling of Proteins with a Flavin Fluorophore.

Bioconjug Chem 2021 08 24;32(8):1559-1563. Epub 2021 Jul 24.

Molecular Enzymology Group, University of Groningen, Nijenborgh 4, Groningen 9747AG, The Netherlands.

Site-specific protein labeling methods are highly valuable tools for research and applications. We present a new protein labeling method that allows covalent attachment of a chromo- and fluorogenic flavin (FMN) to any targeted protein using a short flavinylation peptide-tag. We show that this peptide can be as short as 7 residues and can be located at the N-terminus, C-terminus, or in internal regions of the target protein. Analogous to kinase-catalyzed phosphorylation, the flavin is covalently attached via a stable phosphothreonyl linkage. The site-specific covalent tethering of FMN is accomplished by using a bacterial flavin transferase. The covalent coupling of FMN was shown to work in and cells and could be performed , rendering the "Flavin-tag" method a powerful tool for the selective decoration of proteins with a biocompatible redox-active fluorescent chromophore.
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http://dx.doi.org/10.1021/acs.bioconjchem.1c00306DOI Listing
August 2021

On the diversity of F -dependent oxidoreductases: A sequence- and structure-based classification.

Proteins 2021 11 16;89(11):1497-1507. Epub 2021 Jul 16.

Molecular Enzymology Group, University of Groningen, Groningen, The Netherlands.

The F deazaflavin cofactor is an intriguing molecule as it structurally resembles the canonical flavin cofactor, although behaves as a nicotinamide cofactor due to its obligate hydride-transfer reactivity and similar low redox potential. Since its discovery, numerous enzymes relying on it have been described. The known deazaflavoproteins are taxonomically restricted to Archaea and Bacteria. The biochemistry of the deazaflavoenzymes is diverse and they exhibit great structural variability. In this study a thorough sequence and structural homology evolutionary analysis was performed in order to generate an overarching classification of the F -dependent oxidoreductases. Five different deazaflavoenzyme Classes (I-V) are described according to their structural folds as follows: Class I encompassing the TIM-barrel F -dependent enzymes; Class II including the Rossmann fold F -dependent enzymes; Class III comprising the β-roll F -dependent enzymes; Class IV which exclusively gathers the SH3 barrel F -dependent enzymes and Class V including the three layer ββα sandwich F -dependent enzymes. This classification provides a framework for the identification and biochemical characterization of novel deazaflavoenzymes.
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http://dx.doi.org/10.1002/prot.26170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8518648PMC
November 2021

Modular Assembly of Phosphite Dehydrogenase and Phenylacetone Monooxygenase for Tuning Cofactor Regeneration.

Biomolecules 2021 06 17;11(6). Epub 2021 Jun 17.

Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

The use of multienzyme complexes can facilitate biocatalytic cascade reactions by employing fusion enzymes or protein tags. In this study, we explored the use of recently developed peptide tags that promote complex formation of the targeted proteins: the dimerization-docking and anchoring domain (RIDD-RIAD) system. These peptides allow self-assembly based on specific protein-protein interactions between both peptides and allow tuning of the ratio of the targeted enzymes as the RIAD peptide binds to two RIDD peptides. Each of these tags were added to the C-terminus of a NADPH-dependent Baeyer-Villiger monooxygenase (phenylacetone monooxygenase, PAMO) and a NADPH-regenerating enzyme (phosphite dehydrogenase, PTDH). Several RIDD/RIAD-tagged PAMO and PTDH variants were successfully overproduced in and subsequently purified. Complementary tagged enzymes were mixed and analyzed for their oligomeric state, stability, and activity. Complexes were formed in the case of some specific combinations (PAMO-PTDH and PAMO-PTDH). These enzyme complexes displayed similar catalytic activity when compared with the PTDH-PAMO fusion enzyme. The thermostability of PAMO in these complexes was retained while PTDH displayed somewhat lower thermostability. Evaluation of the biocatalytic performance by conducting conversions revealed that with a self-assembled PAMO-PTDH complex less PTDH was required for the same performance when compared with the PTDH-PAMO fusion enzyme.
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http://dx.doi.org/10.3390/biom11060905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234031PMC
June 2021

Enantioselective oxidation of secondary alcohols by the flavoprotein alcohol oxidase from Phanerochaete chrysosporium.

Arch Biochem Biophys 2021 06 25;704:108888. Epub 2021 Apr 25.

Molecular Enzymology Group, University of Groningen, Nijenborgh 4, Groningen, the Netherlands. Electronic address:

The enantioselective oxidation of secondary alcohols represents a valuable approach for the synthesis of optically pure compounds. Flavoprotein oxidases can catalyse such selective transformations by merely using oxygen as electron acceptor. While many flavoprotein oxidases preferably act on primary alcohols, the FAD-containing alcohol oxidase from Phanerochaete chrysosporium was found to be able to perform kinetic resolutions of several secondary alcohols. By selective oxidation of the (S)-alcohols, the (R)-alcohols were obtained in high enantiopurity. In silico docking studies were carried out in order to substantiate the observed (S)-selectivity. Several hydrophobic and aromatic residues in the substrate binding site create a cavity in which the substrates can comfortably undergo van der Waals and pi-stacking interactions. Consequently, oxidation of the secondary alcohols is restricted to one of the two enantiomers. This study has uncovered the ability of an FAD-containing alcohol oxidase, that is known for oxidizing small primary alcohols, to perform enantioselective oxidations of various secondary alcohols.
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http://dx.doi.org/10.1016/j.abb.2021.108888DOI Listing
June 2021

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties.

J Biol Chem 2021 Jan-Jun;296:100221. Epub 2020 Dec 25.

Molecular Enzymology Group, University of Groningen, Groningen, the Netherlands. Electronic address:

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2'-phosphate being pushed inside the NADP binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.
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http://dx.doi.org/10.1074/jbc.RA120.016297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948450PMC
August 2021

Optimizing the linker length for fusing an alcohol dehydrogenase with a cyclohexanone monooxygenase.

Methods Enzymol 2021 23;647:107-143. Epub 2020 Oct 23.

Molecular Enzymology Group, University of Groningen, Groningen, The Netherlands. Electronic address:

The use of enzymes in organic synthesis is highly appealing due their remarkably high chemo-, regio- and enantioselectivity. Nevertheless, for biosynthetic routes to be industrially useful, the enzymes must fulfill several requirements. Particularly, in case of cofactor-dependent enzymes self-sufficient systems are highly valuable. This can be achieved by fusing enzymes with complementary cofactor dependency. Such bifunctional enzymes are also relatively easy to handle, may enhance stability, and promote product intermediate channeling. However, usually the characteristics of the linker, fusing the target enzymes, are not thoroughly evaluated. A poor linker design can lead to detrimental effects on expression levels, enzyme stability and/or enzyme performance. In this chapter, the effect of the length of a glycine-rich linker was explored for the case study of ɛ-caprolactone synthesis through an alcohol dehydrogenase-cyclohexanone monooxygenase fusion system. The procedure includes cloning of linker variants, expression analysis, determination of thermostability and effect on activity and conversion levels of 15 variants of different linker sizes. The protocols can also be used for the creation of other protein-protein fusions.
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http://dx.doi.org/10.1016/bs.mie.2020.09.008DOI Listing
June 2021

Mutational and structural analysis of an ancestral fungal dye-decolorizing peroxidase.

FEBS J 2021 06 8;288(11):3602-3618. Epub 2021 Jan 8.

Molecular Enzymology Group, University of Groningen, The Netherlands.

Dye-decolorizing peroxidases (DyPs) constitute a superfamily of heme-containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A-C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D-type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D-type DyP ancestor, AncDyPD-b1. Expression of AncDyPD-b1 in Escherichia coli results in large amounts of a heme-containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV-Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady-state kinetics and the crystal structure, reveal fine pH-dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long-range electron transfer process. DATABASE: Structural data are available in the PDB database under the accession number 7ANV.
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http://dx.doi.org/10.1111/febs.15687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248431PMC
June 2021

Production of indigo through the use of a dual-function substrate and a bifunctional fusion enzyme.

Enzyme Microb Technol 2020 Dec 14;142:109692. Epub 2020 Oct 14.

Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG, Groningen, The Netherlands. Electronic address:

The current chemical process for industrial indigo production puts a heavy burden on the environment. An attractive option would be to develop an alternative biotechnological process which does not rely on a petrochemical. This study describes a new biotransformation approach in which l-tryptophan is used as starting material. Its conversion to indigo can be achieved through recombinant overexpression of a bifunctional fusion enzyme, flavin-containing monooxygenase (FMO) fused to tryptophanase (TRP). First, TRP converts l-tryptophan into pyruvate, ammonia and indole. The formed indole serves as substrate for FMO, resulting in indigo formation, while pyruvate fuels the cells for regenerating the required NADPH. To optimize this bioconversion, different fusion constructs were tested. Fusing TRP to FMO at either the N-terminus (TRP-FMO) or the C-terminus (FMO-TRP) resulted in similar high expression levels of bifunctional fusion enzymes. Using whole cells and l-tryptophan as a precursor, high production levels of indigo could be obtained, significantly higher when compared with cells containing only overexpressed FMO. The TRP-FMO containing cells gave the highest yield of indigo resulting in full conversion of 2.0 g l-tryptophan into 1.7 g indigo per liter of culture. The process developed in this study provides an alternative biotransformation approach for the production of indigo starting from biobased starting material.
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http://dx.doi.org/10.1016/j.enzmictec.2020.109692DOI Listing
December 2020

The vast repertoire of carbohydrate oxidases: An overview.

Biotechnol Adv 2021 11 19;51:107634. Epub 2020 Sep 19.

Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG Groningen, the Netherlands. Electronic address:

Carbohydrates are widely abundant molecules present in a variety of forms. For their biosynthesis and modification, nature has evolved a plethora of carbohydrate-acting enzymes. Many of these enzymes are of particular interest for biotechnological applications, where they can be used as biocatalysts or biosensors. Among the enzymes catalysing conversions of carbohydrates are the carbohydrate oxidases. These oxidative enzymes belong to different structural families and use different cofactors to perform the oxidation reaction of CH-OH bonds in carbohydrates. The variety of carbohydrate oxidases available in nature reflects their specificity towards different sugars and selectivity of the oxidation site. Thanks to their properties, carbohydrate oxidases have received a lot of attention in basic and applied research, such that nowadays their role in biotechnological processes is of paramount importance. In this review we provide an overview of the available knowledge concerning the known carbohydrate oxidases. The oxidases are first classified according to their structural features. After a description on their mechanism of action, substrate acceptance and characterisation, we report on the engineering of the different carbohydrate oxidases to enhance their employment in biocatalysis and biotechnology. In the last part of the review we highlight some practical applications for which such enzymes have been exploited.
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http://dx.doi.org/10.1016/j.biotechadv.2020.107634DOI Listing
November 2021

Vanillyl alcohol oxidase.

Enzymes 2020 18;47:87-116. Epub 2020 Jul 18.

Laboratory of Food Chemistry, Wageningen University & Research, Wageningen, The Netherlands. Electronic address:

This review presents a historical outline of the research on vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum, one of the canonical members of the VAO/PCMH flavoprotein family. After describing its discovery and initial biochemical characterization, we discuss the physiological role, substrate scope, and catalytic mechanism of VAO, and review its three-dimensional structure and mechanism of covalent flavinylation. We also explain how protein engineering provided a deeper insight into the role of certain amino acid residues in determining the substrate specificity and enantioselectivity of the enzyme. Finally, we summarize recent computational studies about the migration of substrates and products through the enzyme's structure and the phylogenetic distribution of VAO and related enzymes.
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http://dx.doi.org/10.1016/bs.enz.2020.05.003DOI Listing
December 2020

The multipurpose family of flavoprotein oxidases.

Enzymes 2020 18;47:63-86. Epub 2020 Jul 18.

Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.

This chapter represents a journey through flavoprotein oxidases. The purpose is to excite the reader curiosity regarding this class of enzymes by showing their diverse applications. We start with a brief overview on oxidases to then introduce flavoprotein oxidases and elaborate on the flavin cofactors, their redox and spectroscopic characteristics, and their role in the catalytic mechanism. The six major flavoprotein oxidase families will be described, giving examples of their importance in biology and their biotechnological uses. Specific attention will be given to a few selected flavoprotein oxidases that are not extensively discussed in other chapters of this book. Glucose oxidase, cholesterol oxidase, 5-(hydroxymethyl)furfural (HMF) oxidase and methanol oxidase are four examples of oxidases belonging to the GMC-like flavoprotein oxidase family and that have been shown to be valuable biocatalysts. Their structural and mechanistic features and recent enzyme engineering will be discussed in details. Finally we give a look at the current trend in research and conclude with a future outlook.
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http://dx.doi.org/10.1016/bs.enz.2020.05.002DOI Listing
December 2020

Facile Stereoselective Reduction of Prochiral Ketones by using an F -dependent Alcohol Dehydrogenase.

Chembiochem 2021 01 23;22(1):156-159. Epub 2020 Oct 23.

Molecular Enzymology Group, University of Groningen, Nijenborgh 4, Groningen, The Netherlands.

Effective procedures for the synthesis of optically pure alcohols are highly valuable. A commonly employed method involves the biocatalytic reduction of prochiral ketones. This is typically achieved by using nicotinamide cofactor-dependent reductases. In this work, we demonstrate that a rather unexplored class of enzymes can also be used for this. We used an F -dependent alcohol dehydrogenase (ADF) from Methanoculleus thermophilicus that was found to reduce various ketones to enantiopure alcohols. The respective (S) alcohols were obtained in excellent enantiopurity (>99 % ee). Furthermore, we discovered that the deazaflavoenzyme can be used as a self-sufficient system by merely using a sacrificial cosubstrate (isopropanol) and a catalytic amount of cofactor F or the unnatural cofactor FOP to achieve full conversion. This study reveals that deazaflavoenzymes complement the biocatalytic toolbox for enantioselective ketone reductions.
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http://dx.doi.org/10.1002/cbic.202000651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820951PMC
January 2021

Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence.

J Biol Chem 2020 11 11;295(47):16013-16022. Epub 2020 Sep 11.

Molecular Enzymology Group, University of Groningen, Groningen, The Netherlands. Electronic address:

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.
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http://dx.doi.org/10.1074/jbc.RA120.014996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681015PMC
November 2020

Structure of a robust bacterial protein cage and its application as a versatile biocatalytic platform through enzyme encapsulation.

Biochem Biophys Res Commun 2020 08 15;529(3):548-553. Epub 2020 Jul 15.

Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG, Groningen, the Netherlands. Electronic address:

Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5-9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity.
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http://dx.doi.org/10.1016/j.bbrc.2020.06.059DOI Listing
August 2020

Analysis of the structure and substrate scope of chitooligosaccharide oxidase reveals high affinity for C2-modified glucosamines.

FEBS Lett 2020 09 18;594(17):2819-2828. Epub 2020 Jun 18.

Molecular Enzymology Group, University of Groningen, The Netherlands.

Chitooligosaccharide oxidase (ChitO) is a fungal carbohydrate oxidase containing a bicovalently bound FAD cofactor. The enzyme is known to catalyse the oxidation of chitooligosaccharides, oligomers of N-acetylated glucosamines derived from chitin degradation. In this study, the unique substrate acceptance was explored by testing a range of N-acetyl-d-glucosamine derivatives, revealing that ChitO preferentially accepts carbohydrates with a hydrophobic group attached to C2. The enzyme also accepts streptozotocin, a natural product used to treat tumours. Elucidation of the crystal structure provides an explanation for the high affinity towards C2-decorated glucosamines: the active site has a secondary binding pocket that accommodates groups attached at C2. Docking simulations are fully in line with the observed substrate preference. This work expands the knowledge on this versatile enzyme.
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http://dx.doi.org/10.1002/1873-3468.13854DOI Listing
September 2020

High-level production of industrially relevant oxidases by a two-stage fed-batch approach: overcoming catabolite repression in arabinose-inducible Escherichia coli systems.

Appl Microbiol Biotechnol 2020 Jun 22;104(12):5337-5345. Epub 2020 Apr 22.

Fermentation Pilot Plant, Department of Chemical, Biological and Enviromental Engineering, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain.

With the growing interest in enzyme applications, there is an urgent demand for economic, affordable, and flexible enzyme production processes. In the present paper, we developed a high cell density fed-batch process for the production of two cofactor-containing oxidase, 5-hydroxymethylfurfural oxidase (HMFO) and eugenol oxidase (EUGO). The approach involved the arabinose-inducible system to drive the expression while using mineral media. In order to overcome a major drawback of arabinose-inducible promoters, carbon catabolite repression, (CCR) by glucose, we developed a high cell density culture (HCDC), two-stage fed-batch protocol allowing us to reach cell densities exceeding 70 g/L of dry cell weight (DCW) using glucose as carbon source. Then, induction was achieved by adding arabinose, while changing the carbon source to glycerol. This strategy allowed us to obtain an eightfold increase in recombinant HMFO titer when compared with a reference batch fermentation in Erlenmeyer flasks using terrific broth (TB), typically used with arabinose-inducible strains. The optimized protocol was also tested for expression of a structurally unrelated oxidase, EUGO, where a similar yield was achieved. Clearly, this two-step protocol in which a relatively cheap medium (when compared to TB) can be used reduces costs and provides a way to obtain protein production levels similar to those of IPTG-based systems. KEY POINTS: • Arabinose promoters are not well suited for HCDC production due to CCR effect. • This drawback has been overcome by using a two-stage Fed-batch protocol. • Protein yield has been increased by an eightfold factor, improving process economics.
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http://dx.doi.org/10.1007/s00253-020-10622-yDOI Listing
June 2020

Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.

Elife 2020 03 31;9. Epub 2020 Mar 31.

Molecular Enzymology Group, University of Groningen, Groningen, Netherlands.

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (Δ ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a of 94 °C (+51 °C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.
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http://dx.doi.org/10.7554/eLife.54639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7164962PMC
March 2020

Genome Mining of Oxidation Modules in trans-Acyltransferase Polyketide Synthases Reveals a Culturable Source for Lobatamides.

Angew Chem Int Ed Engl 2020 05 19;59(20):7761-7765. Epub 2020 Mar 19.

Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093, Zurich, Switzerland.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are multimodular megaenzymes that biosynthesize many bioactive natural products. They contain a remarkable range of domains and module types that introduce different substituents into growing polyketide chains. As one such modification, we recently reported Baeyer-Villiger-type oxygen insertion into nascent polyketide backbones, thereby generating malonyl thioester intermediates. In this work, genome mining focusing on architecturally diverse oxidation modules in trans-AT PKSs led us to the culturable plant symbiont Gynuella sunshinyii, which harbors two distinct modules in one orphan PKS. The PKS product was revealed to be lobatamide A, a potent cytotoxin previously only known from a marine tunicate. Biochemical studies show that one module generates glycolyl thioester intermediates, while the other is proposed to be involved in oxime formation. The data suggest varied roles of oxygenation modules in the biosynthesis of polyketide scaffolds and support the importance of trans-AT PKSs in the specialized metabolism of symbiotic bacteria.
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http://dx.doi.org/10.1002/anie.201916005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586987PMC
May 2020

Mechanistic and Crystallographic Studies of Azoreductase AzoA from A01.

ACS Chem Biol 2020 02 29;15(2):504-512. Epub 2020 Jan 29.

GECCO Biotech , Nijenborgh 4 , 9747AG Groningen , The Netherlands.

The azoreductase AzoA from the alkali-tolerant A01 has been studied to reveal its structural and mechanistic details. For this, a recombinant expression system was developed which yields impressive amounts of fully active enzyme. The purified holo enzyme is remarkably solvent-tolerant and thermostable with an apparent melting temperature of 71 °C. The dimeric enzyme contains FMN as a prosthetic group and is strictly NADH dependent. While AzoA shows a negligible ability to use molecular oxygen as an electron acceptor, it is efficient in reducing various azo dyes and quinones. The kinetic and catalytic mechanism has been studied in detail using steady state kinetic analyses and stopped-flow studies. The data show that AzoA performs quinone and azo dye reductions via a two-electron transfer. Moreover, quinones were shown to be much better substrates ( values of 100-400 s for several naphtoquinones) when compared with azo dyes. This suggests that the physiological role of AzoA and sequence-related microbial reductases is linked to quinone reductions and that they can better be annotated as quinone reductases. The structure of AzoA has been determined in complex with FMN at 1.8 Å resolution. AzoA displays unique features in the active site providing clues for explaining its catalytic and thermostability features. An uncommon loop, when compared with sequence-related reductases, forms an active site lid with Trp60 acting as an anchor. Several Trp60 mutants have been analyzed disclosing an important role of this residue in the stability of AzoA, while they retained activity. Structural details are discussed in relation to other azo and quinone reductases. This study provides new insights into the molecular functioning of AzoA and sequence-related reductases.
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http://dx.doi.org/10.1021/acschembio.9b00970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040913PMC
February 2020

Publisher Correction: Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs.

Nat Struct Mol Biol 2020 Feb;27(2):222

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41594-020-0378-8DOI Listing
February 2020

Production of Hydroxy Acids: Selective Double Oxidation of Diols by Flavoprotein Alcohol Oxidase.

Angew Chem Int Ed Engl 2020 03 4;59(12):4869-4872. Epub 2020 Feb 4.

Molecular Enzymology Group, University of Groningen, Nijenborgh 4, Groningen, The Netherlands.

Flavoprotein oxidases can catalyze oxidations of alcohols and amines by merely using molecular oxygen as the oxidant, making this class of enzymes appealing for biocatalysis. The FAD-containing (FAD=flavin adenine dinucleotide) alcohol oxidase from P. chrysosporium facilitated double and triple oxidations for a range of aliphatic diols. Interestingly, depending on the diol substrate, these reactions result in formation of either lactones or hydroxy acids. For example, diethylene glycol could be selectively and fully converted into 2-(2-hydroxyethoxy)acetic acid. Such a facile cofactor-independent biocatalytic route towards hydroxy acids opens up new avenues for the preparation of polyester building blocks.
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http://dx.doi.org/10.1002/anie.201914877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079103PMC
March 2020

Biocatalytic Enantioselective Oxidation of -Allylic Alcohols with Flavin-Dependent Oxidases.

Adv Synth Catal 2019 Nov 10;361(22):5264-5271. Epub 2019 Oct 10.

Institute of Chemistry, NAWI Graz, BioTechMed Graz University of Graz Heinrichstr. 28 8010 Graz Austria.

The oxidation of allylic alcohols is challenging to perform in a chemo- as well as stereo-selective fashion at the expense of molecular oxygen using conventional chemical protocols. Here, we report the identification of a library of flavin-dependent oxidases including variants of the berberine bridge enzyme (BBE) analogue from (BBE15) and the 5-(hydroxymethyl)furfural oxidase (HMFO) and its variants (V465T, V465S, V465T/W466H and V367R/W466F) for the enantioselective oxidation of -allylic alcohols. While and benzylic alcohols as well as certain sugars are well known to be transformed by flavin-dependent oxidases, -allylic alcohols have not been studied yet except in a single report. The model substrates investigated were oxidized enantioselectively in a kinetic resolution with an E-value of up to >200. For instance HMFO V465S/T oxidized the ()-enantiomer of ()-oct-3-en-2-ol () and ()-4-phenylbut-3-en-2-ol with E>200 giving the remaining ()-alcohol with >99% at 50% conversion. The enantioselectivity could be decreased if required by medium engineering by the addition of cosolvents (e. g. dimethyl sulfoxide).
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http://dx.doi.org/10.1002/adsc.201900921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919931PMC
November 2019

Creating Flavin Reductase Variants with Thermostable and Solvent-Tolerant Properties by Rational-Design Engineering.

Chembiochem 2020 05 21;21(10):1481-1491. Epub 2020 Feb 21.

School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), 555 Moo 1 Payupnai, Wangchan, Rayong, 21210, Thailand.

We have employed computational approaches-FireProt and FRESCO-to predict thermostable variants of the reductase component (C ) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6-5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C variants remain active and generate reduced flavin mononucleotide (FMNH ) for reactions catalyzed by bacterial luciferase and by the monooxygenase C more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300-500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C enzyme can lead to broad-spectrum uses of C as a redox biocatalyst for future industrial applications.
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http://dx.doi.org/10.1002/cbic.201900737DOI Listing
May 2020

Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs.

Nat Struct Mol Biol 2020 01 23;27(1):14-24. Epub 2019 Dec 23.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.

Flavin-containing monooxygenases (FMOs) are ubiquitous in all domains of life and metabolize a myriad of xenobiotics, including toxins, pesticides and drugs. However, despite their pharmacological importance, structural information remains bereft. To further our understanding behind their biochemistry and diversity, we used ancestral-sequence reconstruction, kinetic and crystallographic techniques to scrutinize three ancient mammalian FMOs: AncFMO2, AncFMO3-6 and AncFMO5. Remarkably, all AncFMOs could be crystallized and were structurally resolved between 2.7- and 3.2-Å resolution. These crystal structures depict the unprecedented topology of mammalian FMOs. Each employs extensive membrane-binding features and intricate substrate-profiling tunnel networks through a conspicuous membrane-adhering insertion. Furthermore, a glutamate-histidine switch is speculated to induce the distinctive Baeyer-Villiger oxidation activity of FMO5. The AncFMOs exhibited catalysis akin to human FMOs and, with sequence identities between 82% and 92%, represent excellent models. Our study demonstrates the power of ancestral-sequence reconstruction as a strategy for the crystallization of proteins.
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http://dx.doi.org/10.1038/s41594-019-0347-2DOI Listing
January 2020
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