BBE-like enzymes

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Berberine bridge enzyme-like (BBE-like enzymes) form a subgroup of the superfamily of FAD-linked oxidases (SCOPe d.58.32), structurally characterized by a typical fold observed initially for vanillyl-alcohol oxidase (VAO). [1] This proteins are part of a multigene family (PF08031) that can be found in plants, fungi and bacteria. [2]

Contents

BBE-like enzymes family form a large subgroup that have a special C-terminal structural element adjacent to the substrate binding region. An homonym of this family is the (S)-reticuline oxidase or berberine bridge enzyme from California poppy (Eschscholzia californica), the responsible of catalyzing the conversion of (S)-reticuline to (S)-scoulerine. This conversion is made by an oxidative ring closure reaction. The product of this reaction is the C-C bond and is referred to as the berberine bridge. Also, marks a branch point in the biosynthesis of benzylisoquinoline alkaloids.

As mentioned above, BBE-like enzymes are in the large family of FAD-linked oxidases. Regarding the structure of this particular family, they have a FAD binding module formed by the N- and C- terminal parts of the protein. There is a substrate binding module that, in collaboration with isoalloxazine ring of FAD, disposes the environment for efficient substrate binding and oxidation. [3]

A: Structural properties of the BBE-like enzymes. Sub-domains of the upper flavin-binding module are shown in purple, blue and red. B: Secondary structure elements are in consecutively order for a-helices and b-strands in the flavin- and substrate-binding modules and are differenced in green and blue, respectively. BBE-like enzyme structure.jpg
A: Structural properties of the BBE-like enzymes. Sub-domains of the upper flavin-binding module are shown in purple, blue and red. B: Secondary structure elements are in consecutively order for a-helices and b-strands in the flavin- and substrate-binding modules and are differenced in green and blue, respectively.

Purification process

Cell cultures of Berberis beaniana (B. beaniana), which in this certain experiment are taken as an example, were harvested 10–12 days, contained large amounts of proto-berberines, mainly jatrorrhizine. These quaternary alkaloids have a strong inhibitory effect on the BBE, so they had to be removed. In order to eliminate most of these interfering cationic substances, the enzyme solution was treated first with carboxymethyl-Sepharose and subsequently with dextrancoated charcoal.

After that, the resulting solution was fractionated using standard procedures and generated, after isoelectric focusing, a single protein band in SDS gel electrophoresis. In the end, the obtained enzyme had been purified 450 times and contained 0.7% of the activity present in the crude extract at the beginning. [4]

Properties

pH scale showing the BBE-like enzyme's optimum pH and isoelectric point. 141128 Phil Holliday - pH scale final.png
pH scale showing the BBE-like enzyme's optimum pH and isoelectric point.

The BBE-like enzyme's optimum pH is 8.9, this means it works in an alkaline medium, but the isoelectric point of the homogeneous enzyme is located at pH 4.9, which is a rather an acid medium. This value was obtained with isoelectric focusing and chromatofocusing techniques.

The enzyme broad temperature range is between 40-50 degrees Celsius. The molecular weight of the protein was determined by two processes that show two different results: by SDS gel electrophoresis comes to be 54 kD, and by gel filtration on AcA 54 the enzyme corresponds to a molecular weight of 49 kD, adopting globular shape. A purification study shows that the true molecular weight is in the range of 52 ±4 kD.

On the other hand, total activity decreases drastically during the stationary phase. [4]

Known functions

BBE-like enzymes serve as a catalyzer for a wide range of reactions. All the way from two-electron oxidations as observed in (At)BBE-like 15 to four-electron oxidations as seen in Dbv29.

BBE-like enzymes are involved in the synthesis of plenty of isoquinoline alkaloids such as the conversion of (S)-reticuline to (S)-scoulerine [5] or by guiding (S)-reticuline to protoberberine, protopine, benzophenanthridine, phthalide isoquinoline or rhoeadine metabolic pathways. [4]

Research on this matter is rare since it is very complex and had never been looked into until recently. New studies reveal that BBE-like enzymes are involved in the biological synthesis of the alkaloids intermediates communesin as well as chanoclavine (I). The mechanism through which reactions are catalyzed by these BBE-like enzymes has not been found yet, but the resulting conformation of the products suggests that a similar coupling of substrate oxidation and ring formation occurs in these processes.

BBE-like enzymes in plants

BBE-like enzymes in plant physiology play a role in primary metabolism catalyzed by members of this family. The oxidation of a variety of alcohol groups supplies a first understanding of the origin of the BBE-like enzyme family, present in reactions like oxidation of mono and polysaccharides. It seems that they are present in a huge percentage of plants.

A common reaction of BBE-like enzymes from plants is the oxidation of carbohydrates at the anomeric center to the appropriate lactones. A member of these enzymes is hexose oxidase (HOX) from Chondrus crispus, a red algae that belongs to the division of Rhodophyta. But is more related to bacterial BBE-like enzymes than to members present in plants.

Other example of carbohydrate oxidizing BBE-like enzyme is nectarin V (Nec5) from tobacco. Its function is to convert glucose to gluconolactone. Nec5 is related with the pathogen defense system of plants, because it protects reproductive organelles. [3]

Another function of this class of enzymes in plants is Defense response homeostasis. BBE-like enzymes play a key role as oligosaccharide oxidases, reducing the activity of olygogalacturonanes (fragments of pectin) [6] and oligocellobiose (fragments of cellulose) [7] as DAMPS.

Medical applications

Berberine bridge enzyme (BBE) is a central enzyme in the biosynthesis of berberine, a pharmaceutically important alkaloid. The enzyme itself hasn't had extensive research carried on, and has very limited, if any, specific medical application. On the other hand, berberine is highly regarded for its interactions with several diseases. Berberine has been known to influence weight loss, and this antiobesity effect may benefit all conditions related to increased body mass such as hypertension, dyslipidemia or pre-diabetes. This may reduce the likelihood of getting sedentary diseases such as heart failure or other problems related to this issue. By being an AMPK activator like Metformin, it acts similar, affecting metabolism in a way that may reveal useful applications to treat type-2 diabetes.

Overall, this alkaloid might be useful in the treatment and study of diseases like polycystic ovary syndrome (PCOS), some types of cancer, heart problems or dyslipidemia. [8]

Brief history

First discovered and named by E. Rink and H. Böhm in 1974 they were isolated in cell suspension cultures of the Papaveraceae Macleaya microcarpa. [9]

Since then, many minor advancements have been made in this particular topic, but BBE-like enzymes, either for their lack of study, or the lack of clear indications that it might contain useful information for other scientific research, have yet to be fully worked on, and subsequently known at their full complexity.

Related Research Articles

A dehydrogenase is an enzyme belonging to the group of oxidoreductases that oxidizes a substrate by reducing an electron acceptor, usually NAD+/NADP+ or a flavin coenzyme such as FAD or FMN. Like all catalysts, they catalyze reverse as well as forward reactions, and in some cases this has physiological significance: for example, alcohol dehydrogenase catalyzes the oxididation of ethanol to acetaldehyde in animals, but in yeast it catalyzes the production of ethanol from acetaldehyde.

Flavin adenine dinucleotide Redox-active coenzyme

In biochemistry, flavin adenine dinucleotide (FAD) is a redox-active coenzyme associated with various proteins, which is involved with several enzymatic reactions in metabolism. A flavoprotein is a protein that contains a flavin group, which may be in the form of FAD or flavin mononucleotide (FMN). Many flavoproteins are known: components of the succinate dehydrogenase complex, α-ketoglutarate dehydrogenase, and a component of the pyruvate dehydrogenase complex.

Noscapine Chemical compound

Noscapine is a benzylisoquinoline alkaloid, of the phthalideisoquinoline structural subgroup, which has been isolated from numerous species of the family Papaveraceae. It lacks significant hypnotic, euphoric, or analgesic effects affording it with very low addictive potential. This agent is primarily used for its antitussive (cough-suppressing) effects.

Sanguinarine

Sanguinarine is a polycyclic quaternary alkaloid. It is extracted from some plants, including the bloodroot plant, from whose taxonomic name, Sanguinaria canadensis, its name is drawn; the Mexican prickly poppy ; Chelidonium majus; and Macleaya cordata.

Scoulerine Chemical compound

Scoulerine, also known as discretamine and aequaline, is a benzylisoquinoline alkaloid (BIA) that is derived directly from (S)-reticuline through the action of berberine bridge enzyme. It is a precursor of other BIAs, notably berberine, noscapine, (S)-tetrahydropalmatine, and (S)-stylopine, as well as the alkaloids protopine, and sanguinarine. It is found in many plants, including opium poppy, Croton flavens, and certain plants in the genus Erythrina.

Pectinesterase Class of enzymes

Pectinesterase (PE) is a ubiquitous cell-wall-associated enzyme that presents several isoforms that facilitate plant cell wall modification and subsequent breakdown. It is found in all higher plants as well as in some bacteria and fungi. Pectinesterase functions primarily by altering the localised pH of the cell wall resulting in alterations in cell wall integrity.

Berberine Quaternary ammonium cation

Berberine is a quaternary ammonium salt from the protoberberine group of benzylisoquinoline alkaloids found in such plants as Berberis, such as Berberis vulgaris (barberry), Berberis aristata, Mahonia aquifolium, Hydrastis canadensis (goldenseal), Xanthorhiza simplicissima (yellowroot), Phellodendron amurense, Coptis chinensis, Tinospora cordifolia, Argemone mexicana, and Eschscholzia californica. Berberine is usually found in the roots, rhizomes, stems, and bark.

Translocase is a general term for a protein that assists in moving another molecule, usually across a cell membrane. These enzymes catalyze the movement of ions or molecules across membranes or their separation within membranes. The reaction is designated as a transfer from “side 1” to “side 2” because the designations “in” and “out”, which had previously been used, can be ambiguous. Translocases are the most common secretion system in Gram positive bacteria.

(S)-Tetrahydroberberine oxidase is an enzyme that catalyzes the final transformation in the biosynthesis of berberine, a quaternary benzylisoquinoline alkaloid of the protoberberine structural subgroup. This reaction pathway catalyzes the four-electron oxidation of (S)-tetrahydroberberine in the presence of oxygen to produce berberine and hydrogen peroxide as products.

In enzymology, a columbamine oxidase (EC 1.21.3.2) is an enzyme that catalyzes the chemical reaction

In enzymology, a reticuline oxidase (EC 1.21.3.3) is an enzyme that catalyzes the chemical reaction

In enzymology, a (S)-cheilanthifoline synthase (EC 1.14.21.2) is an enzyme that catalyzes the chemical reaction

Vanillyl-alcohol oxidase

In enzymology, a vanillyl-alcohol oxidase (EC 1.1.3.38) is an enzyme that catalyzes the chemical reaction

L-amino-acid oxidase

In enzymology, an L-amino acid oxidase (LAAO) (EC 1.4.3.2) is an enzyme that catalyzes the chemical reaction

Proline dehydrogenase

In enzymology, proline dehydrogenase (PRODH) is an enzyme of the oxidoreductase family, active in the oxidation of L-proline to (S)-1-pyrroline-5-carboxylate during proline catabolism. The end product of this reaction is then further oxidized in a (S)-1-pyrroline-5-carboxylate dehydrogenase (P5CDH)-dependent reaction of the proline metabolism, or spent to produce ornithine, a crucial metabolite of ornithine and arginine metabolism. The systematic name of this enzyme class is L-proline:quinone oxidoreductase. Other names in common use include L-proline dehydrogenase, L-proline oxidase,and L-proline:(acceptor) oxidoreductase. It employs one cofactor, FAD, which requires riboflavin.

Canadine Chemical compound

(S)-Canadine, also known as (S)-tetrahydroberberine and xanthopuccine, is a benzylisoquinoline alkaloid (BIA), of the protoberberine structural subgroup, and is present in many plants from the family Papaveraceae, such as Corydalis yanhusuo and C. turtschaninovii.

Protopine Chemical compound

Protopine is an alkaloid occurring in opium poppy, Corydalis tubers and other plants of the family papaveraceae, like Fumaria officinalis. Protopine is metabolically derived from the benzylisoquinoline alkaloid (S)-Reticuline through a progressive series of five enzymatic transformations: 1) berberine bridge enzyme to (S)-Scoulerine; 2) (S)-cheilanthifoline synthase/CYP719A25 to (S)-Cheilanthifoline; 3) (S)-stylopine synthase/CYP719A20 to (S)-Stylopine; 4) (S)-tetrahydroprotoberberine N-methyltransferase to (S)-cis-N-Methylstylopine; and ultimately, 5) N-methylstylopine hydroxylase to protopine.

Tetrahydrocannabinolic acid synthase

Tetrahydrocannabinolic acid (THCA) synthase is an enzyme responsible for catalyzing the formation of THCA from cannabigerolic acid (CBGA). THCA is the direct precursor of tetrahydrocannabinol (THC), the principal psychoactive component of cannabis, which is produced from various strains of Cannabis sativa. Therefore, THCA synthase is considered to be a key enzyme controlling cannabis psychoactivity. Polymorphisms of THCA synthase result in varying levels of THC in Cannabis plants, resulting in "drug-type" and "fiber-type" C. sativa varieties.

Cannabidiolic acid synthase is an enzyme with systematic name cannabigerolate:oxygen oxidoreductase . It is an oxidoreductase found in Cannabis sativa that catalyses the formation of cannabidiolate, a carboxylated precursor of cannabidiol.

Salutaridinol Chemical compound

Salutaridinol is a modified benzyltetrahydroisoquinoline alkaloid with the formula C19H23NO4. It is produced in the secondary metabolism of the opium poppy Papaver somniferum (Papaveraceae) as an intermediate in the biosynthetic pathway that generates morphine. As an isoquinoline alkaloid, it is fundamentally derived from tyrosine as part of the shikimate pathway of secondary metabolism. Salutaridinol is a product of the enzyme salutaridine: NADPH 7-oxidoreductase and the substrate for the enzyme salutaridinol 7-O-acetyltransferase, which are two of the four enzymes in the morphine biosynthesis pathway that generates morphine from (R)-reticuline. Salutaridinol's unique position adjacent to two of the four enzymes in the morphine biosynthesis pathway gives it an important role in enzymatic, genetic, and synthetic biology studies of morphine biosynthesis. Salutaridinol levels are indicative of the flux through the morphine biosynthesis pathway and the efficacy of both salutaridine: NADPH 7-oxidoreductase and salutaridinol 7-O-acetyltransferase.

References

  1. ↑ Mattevi, A.; Fraaije, M. W.; Mozzarelli, A.; Olivi, L.; Coda, A.; van Berkel, W. J. H. (1997-07-15). "Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity" (PDF). Structure. 5 (7): 907–920. doi:10.1016/S0969-2126(97)00245-1. PMID   9261083. S2CID   36071420.
  2. ↑ Daniel B, Wallner S, Steiner B, Oberdorfer G, Kumar P, van der Graaff E, et al. (2016) Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae. PLoS ONE 11(6): e0156892. doi:10.1371/journal. pone.0156892
  3. 1 2 3 Daniel B, Konrad B, Toplak M, Lahham M, Messenlehner J, Winkler A, Macheroux P., "The family of berberine bridge enzyme-like enzymes: A treasure-trove of oxidative reactions" Archives of Biochemistry and Biophysics (2017), https://dx.doi.org/10.1016/j.abb.2017.06.023
  4. 1 2 3 Stevens, P.; Naotaka N.; Meinhart H.Z. Purification and characterization of the berberine bridge enzyme from berberis beaniana cell cultures Phytochemistry, Vol. 24, No. 11, pp. 2577-2583, (1985).
  5. ↑ Andreas W., Franz H., Toni M. K., Anton G., and Peter M. Biochemical Evidence That Berberine Bridge Enzyme Belongs to a Novel Family of Flavoproteins Containing a Bi-covalently Attached FAD Cofactor. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 30, pp. 21276 –21285, July 28, 2006
  6. ↑ Benedetti, Manuel; Verrascina, Ilaria; Pontiggia, Daniela; Locci, Federica; Mattei, Benedetta; De Lorenzo, Giulia; Cervone, Felice (April 2018). "Four Arabidopsis berberine bridge enzyme-like proteins are specific oxidases that inactivate the elicitor-active oligogalacturonides". The Plant Journal. 94 (2): 260–273. doi:10.1111/tpj.13852. PMID   29396998. S2CID   4531290.
  7. ↑ Locci, Federica; Benedetti, Manuel; Pontiggia, Daniela; Citterico, Matteo; Caprari, Claudio; Mattei, Benedetta; Cervone, Felice; De Lorenzo, Giulia (May 2019). "An Arabidopsis berberine bridge enzyme‐like protein specifically oxidizes cellulose oligomers and plays a role in immunity". The Plant Journal. 98 (3): 540–554. doi:10.1111/tpj.14237. ISSN   0960-7412. PMID   30664296. S2CID   58557174.
  8. ↑ "Clinical Applications for Berberine". Natural Medicine Journal. Retrieved 2017-10-18.
  9. ↑ Rink E. and Böhm H.(1975), Conversion of reticuline into scoulerine by a cell free preparation from Macleaya microcarpa cell suspension cultures, FEBS Letters, 49, doi: 10.1016/0014-5793(75)80794-0
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