Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the ...biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family.
Berberine bridge enzyme-like proteins are a multigene family in plants.
Members of the berberine bridge enzyme-like family were identified as monolignol oxidoreductases.
Berberine bridge enzyme-like enzymes play a role in monolignol metabolism and lignin formation.
Our results indicate a novel and unexpected role of berberine bridge enzyme-like enzymes in plant biochemistry and physiology.
Hydratases provide access to secondary and tertiary alcohols by regio‐ and/or stereospecifically adding water to carbon‐carbon double bonds. Thereby, hydroxy groups are introduced without the need ...for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure‐based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two‐electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio‐ and stereoselective hydration.
We report the first crystal structure of an oleate hydratase in complex with the essential flavin cofactor. Elucidation of the active‐site amino acid residues allowed us to propose a structure‐based reaction mechanism for this class of hydratases that accounts for the role of the flavin moiety. Our findings should help employment of this enzyme class in future biocatalytic applications.
Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly ...show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.
The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction ...(dehydrogenases). Previously, a ‘gatekeeper’ amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60 000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the ‘oxyanion hole’ in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE‐like enzymes. Analysis of flavin‐dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle. DATABASE: Structural data are available in PDB database under the accession numbers 4PVE, 4PVH, 4PVJ, 4PVK, 4PWB, 4PWC and 4PZF .
A chemoenzymatic approach for the asymmetric total synthesis of the title compounds is described that employs an enantioselective oxidative C–C bond formation catalyzed by berberine bridge enzyme ...(BBE) in the asymmetric key step. This unique reaction yielded enantiomerically pure (R)-benzylisoquinoline derivatives and (S)-berbines such as the natural product (S)-scoulerine, a sedative and muscle relaxing agent. The racemic substrates rac-1 required for the biotransformation were prepared in 4–8 linear steps using either a Bischler–Napieralski cyclization or a C1–Cα alkylation approach. The chemoenzymatic synthesis was applied to the preparation of fourteen enantiomerically pure alkaloids, including the natural products (S)-scoulerine and (R)-reticuline, and gave overall yields of up to 20% over 5–9 linear steps.
Berberine bridge enzyme (BBE) is a paradigm for the class of bicovalently flavinylated oxidases, which catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine. His174 was identified ...as an important active site residue because of its role in the stabilization of the reduced state of the flavin cofactor. It is also strictly conserved in the family of BBE-like oxidases. Here, we present a detailed biochemical and structural characterization of a His174Ala variant supporting its importance during catalysis and for the structural organization of the active site. Substantial changes in all kinetic parameters and a decrease in midpoint potential were observed for the BBE His174Ala variant protein. Moreover, the crystal structure of the BBE His174Ala variant showed significant structural rearrangements compared to wild-type enzyme. On the basis of our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1–C2O locus via interaction with the hydroxyl group at C2′ of the ribityl side chain of the flavin cofactor. Hence, replacement of this residue with alanine reduces the stabilizing effect for the transiently formed negative charge and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential.
Anthranoyl-CoA monooxygenase/reductase (ACMR) participates in an unusual pathway for the degradation of aromatic compounds in Azoarcus evansii. It catalyzes the monooxygenation of anthranoyl-CoA to ...5-hydroxyl-2-aminobenzoyl-CoA and the subsequent reduction to the dearomatized product 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. The two reactions occur in separate domains, termed the monooxygenase and reductase domain. Both domains were reported to utilize FAD as a cofactor for hydroxylation and reduction, respectively. We have heterologously expressed ACMR in Escherichia coli BL21 and found that the monooxygenase domain contains FAD. However, the reductase domain utilizes FMN and not FAD for the reduction of the intermediate 5-hydroxyl-2-aminobenzoyl-CoA. A homology model for the reductase domain predicted a topology similar to the Old Yellow Enzyme family, which exclusively bind FMN, in accordance with our results. Binding studies with 2-aminobenzoyl-CoA (AbCoA) and p-hydroxybenzaldehyde (pHB) as probes for the monooxygenase and reductase domain, respectively, indicated that two functionally distinct and independent active sites exist. Given the homodimeric quartenary structure of ACMR and the compact shape of the dimer as determined by small-angle X-ray scattering experiments we propose that the monooxygenase and reductase domain of opposite peptide chains are involved in the transformation of anthranoyl-CoA to 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA.
•Successful heterologous expression of soluble anthranoyl-CoA monooxygenase/reductase from Azoarcus evansii•Identification of FMN and FAD as cofactors for the reductase and monooxygenase domain, respectively•The reductase domain adopts an (αβ)8-barrel topology and belongs to the Old Yellow Enzyme family
The subfamily of sarcosine oxidase is a set of enzymes within the larger family of amine oxidases. It is ubiquitously distributed among different kingdoms of life. The member enzymes catalyze the ...oxidization of an N-methyl amine bond of amino acids to yield unstable imine species that undergo subsequent spontaneous non-enzymatic reactions, forming an array of different products. These products range from demethylated simple species to complex alkaloids. The enzymes belonging to the sarcosine oxidase family, namely, monomeric and heterotetrameric sarcosine oxidase, l-pipecolate oxidase, N-methyltryptophan oxidase, NikD, l-proline dehydrogenase, FsqB, fructosamine oxidase and saccharopine oxidase have unique features differentiating them from other amine oxidases. This review highlights the key attributes of the sarcosine oxidase family enzymes, in terms of their substrate binding motif, type of oxidation reaction mediated and FAD regeneration, to define the boundaries of this group and demarcate these enzymes from other amine oxidase families.
Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are utilized as coenzymes in many biochemical reduction-oxidation reactions owing to the ability of the tricyclic isoalloxazine ring ...system to employ the oxidized, radical and reduced state. We have analyzed the genome of Arabidopsis thaliana to establish an inventory of genes encoding flavin-dependent enzymes (flavoenzymes) as a basis to explore the range of flavin-dependent biochemical reactions that occur in this model plant. Expectedly, flavoenzymes catalyze many pivotal reactions in primary catabolism, which are connected to the degradation of basic metabolites, such as fatty and amino acids as well as carbohydrates and purines. On the other hand, flavoenzymes play diverse roles in anabolic reactions most notably the biosynthesis of amino acids as well as the biosynthesis of pyrimidines and sterols. Importantly, the role of flavoenzymes goes much beyond these basic reactions and extends into pathways that are equally crucial for plant life, for example the production of natural products. In this context, we outline the participation of flavoenzymes in the biosynthesis and maintenance of cofactors, coenzymes and accessory plant pigments (e. g. carotenoids) as well as phytohormones. Moreover, several multigene families have emerged as important components of plant immunity, for example the family of berberine bridge enzyme-like enzymes, flavin-dependent monooxygenases and NADPH oxidases. Furthermore, the versatility of flavoenzymes is highlighted by their role in reactions leading to tRNA-modifications, chromatin regulation and cellular redox homeostasis. The favorable photochemical properties of the flavin chromophore are exploited by photoreceptors to govern crucial processes of plant adaptation and development. Finally, a sequence- and structure-based approach was undertaken to gain insight into the catalytic role of uncharacterized flavoenzymes indicating their involvement in unknown biochemical reactions and pathways in A. thaliana.
Display omitted
•262 genes encode flavoproteins accounting for 0.93% of the protein coding regions.•The genome harbors a remarkable number of multigene families of flavoenzymes.•Flavoenzymes are crucial for plant-specific reactions in secondary metabolism.•Flavoproteins play central roles in processes regulated by blue light.•As many as 37 putative flavoenzymes are involved in unknown reactions.
Monolignol oxidoreductases are members of the berberine bridge enzyme–like (BBE-like) protein family (pfam 08031) that oxidize monolignols to the corresponding aldehydes. They are FAD-dependent ...enzymes that exhibit the para-cresolmethylhydroxylase-topology, also known as vanillyl oxidase-topology. Recently, we have reported the structural and biochemical characterization of two monolignol oxidoreductases from Arabidopsis thaliana, AtBBE13 and AtBBE15. Now, we have conducted a comprehensive site directed mutagenesis study for AtBBE15, to expand our understanding of the catalytic mechanism of this enzyme class. Based on the kinetic properties of active site variants and molecular dynamics simulations, we propose a refined, structure-guided reaction mechanism for the family of monolignol oxidoreductases. Here, we propose that this reaction is facilitated stepwise by the deprotonation of the allylic alcohol and a subsequent hydride transfer from the Cα-atom of the alkoxide to the flavin. We describe an excessive hydrogen bond network that enables the catalytic mechanism of the enzyme. Within this network Tyr479 and Tyr193 act concertedly as active catalytic bases to facilitate the proton abstraction. Lys436 is indirectly involved in the deprotonation as this residue determines the position of Tyr193 via a cation-π interaction. The enzyme forms a hydrophilic cavity to accommodate the alkoxide intermediate and to stabilize the transition state from the alkoxide to the aldehyde. By means of molecular dynamics simulations, we have identified two different and distinct binding modes for the substrate in the alcohol and alkoxide state. The alcohol interacts with Tyr193 and Tyr479 while Arg292, Gln438 and Tyr193 form an alkoxide binding site to accommodate this intermediate. The pH-dependency of the activity of the active site variants revealed that the integrity of the alkoxide binding site is also crucial for the fine tuning of the pKa of Tyr193 and Tyr479. Sequence alignments showed that key residues for the mechanism are highly conserved, indicating that our proposed mechanism is not only relevant for AtBBE15 but for the majority of BBE-like proteins.
Display omitted
PDF
