Biocatalytic Organic Synthesis of Optically Pure (<i>S</i>)-Scoulerine and Berbine and Benzylisoquinoline Alkaloids

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 (<i>R</i>)-benzylisoquinoline derivatives and (<i>S</i>)-berbines such as the natural product (<i>S</i>)-scoulerine, a sedative and muscle relaxing agent. The racemic substrates <i>rac</i>-<b>1</b> 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 (<i>S</i>)-scoulerine and (<i>R</i>)-reticuline, and gave overall yields of up to 20% over 5–9 linear steps

Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

<div><p>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 <i>Arabidopsis thaliana</i> 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 <i>At</i>BBE-like protein family (termed <i>At</i>BBE-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 <i>At</i>BBE-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 <i>At</i>BBE-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 <i>At</i>BBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.</p></div

<i>At</i>BBE-like 28 loss-of-function affects biomass formation and salt stress tolerance.

<p>A: The <i>bbe28</i> mutant forms less biomass compared to Col-0, based on fresh weight (FW, black bars) and dry weight (DW, grey bars). B: The <i>bbe28</i> mutant is more sensitive to salt stress compared to Col-0. Black bars control medium, grey bars salt stress medium (100 mM NaCl). Average values ± SE are shown, with relative biomass (%) compared to Col-0 in A and percentage of green healthy shoots vs. total number seedlings in B. * indicates the statistically significant difference (Student’s t-test) when compared to Col-0 at p<0.05.</p

A: Overall topology (A) and active site forming secondary elements (B) of <i>At</i>BBE-like 28.

<p>A: Green: FAD-binding domain, orange: substrate-binding domain. The active site is formed between the FAD-binding site and a substrate binding domain B: structural elements interacting directly with the isoalloxazine ring of the FAD-cofactor and structural elements harboring residues involved in the formation of the active site.</p

Overlay of <i>At</i>BBE-like 28 and Phl p 4 I153V N158H.

<p>A: <i>re</i>-side: <i>At</i>BBE-like 28 is shown in green and yellow, Phl p4 (PDB: 4PWC) is shown in magenta. The chloride ion embedded in <i>At</i>BBE-like 28 is shown in green, bromide from Phl p 4 is shown in red. The oxygen pocket is highly conserved and in both structures occupied by oxygen surrogating halide ions. In Phl p4 the halide ion is complexed by the nitrogen of the peptide bond between Cys150 and Val149 and the nitrogen of the peptide bond between Val149 and Gly148. In <i>At</i>BBE-like 28 the halide ion is complexed by the corresponding residues, additionally His174 and Gln182 are involved in hydrogen bonds towards the chloride ion. B: <i>si</i>-side: The positions of the active site forming residues are conserved. Their nature has been changed, leading to two different active sites embedded in a very similar protein scaffold. Though the position of the isoalloxazine ring is highly conserved in the BBE-like family the C4 C6 axis of the plane N10 C4 C6 has been shifted 32° resulting in a displacement of N5 by 1.6 Å.</p

Phylogenetic tree of the BBE-like enzymes within the <i>Brassicaceae</i> familiy.

<p>Sequences from <i>Arabidopsis lyrata</i> (<i>Al</i>), <i>Arabidopsis thaliana</i> (<i>At</i>), <i>Boechera stricta</i> (<i>Bs</i>), <i>Brassica rapa</i> (<i>Br</i>), <i>Capsella grandiflora</i> (<i>Cg</i>), <i>Capsella rubella</i> (<i>Cr</i>), and <i>Eutrema salsugineum</i> (<i>Es</i>) were used. The composition of the phylogenetic groups is summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156892#pone.0156892.t001" target="_blank">Table 1</a>.</p

Active site composition of types I-IV (panel A to D) of BBE-like enzymes.

<p>The isoalloxazine ring is shown in yellow. Residues forming the active site are shown in green and residues involved in proton abstraction during catalysis are shown in violet. A: Type I: Tyr117 and Gln438 are engaged in a hydrogen bond. Tyr193 acts as catalytic base, it is stabilized in the deprotonated state by Tyr479 and Lys436 (PDB entry 4UD8). B: Type II. Tyr113 and Gln428 are engaged in a hydrogen bond. Tyr188 putatively acts as catalytic base after de-protonation by Glu426 (PDB entry 5D79). C: Type III: Glu430 putatively acts as catalytic base in the active site. The involvement of Tyr472 and Tyr185 is conceivable. A homology model of Q9SA88 based on 4UD8 and prepared with YASARA was used for visualization [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156892#pone.0156892.ref014" target="_blank">14</a>]. D: Type IV: Tyr88 and Gln399 are engaged in a hydrogen bond, Tyr439 acts as catalytic base after de-protonation by Asp372 via a water molecule (PDB entry 4PWC).</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Sequence logos representing the secondary elements directly interacting with the isoalloxazine ring.

<p>The numbering is according to the sequence of <i>At</i>BBE-like 28. *: site of covalent cofactor attachment, ▼: Gatekeeper residue controlling access to the oxygen pocket; valine is found in oxidases, leucine in dehydrogenases. Black boxes: Variable polar residues putatively involved in catalysis. Red boxes: Variable aromatic residues putatively involved in catalysis. β14, 16, 15 and 11 cover the <i>si</i>-side of the isoalloxazine ring. Arrows indicate the orientation of the residues in the β-strands. An upward arrow indicates the residue points towards the α-helices covering the 7-stranded antiparallel β-sheet (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156892#pone.0156892.g002" target="_blank">Fig 2B</a>). These residues are found to be structurally relevant and are predominantly conserved in the overall family (compare β14 positions 366, 368, 370 and β16 position 433). A downward arrow indicates the residue points towards the active site. Residues contributing to the decoration of the active site are conserved within the phylogenetic groups but are variable within the overall family (compare β16 positions 426, 428 and β15 position 401).</p

Model of the active site of <i>At</i>BBE-like 28 indicating the putative site of oxidative. attack (red sphere).

<p>Interactions are represented by dashed yellow lines. One of the unique features of the type IIa active site is the nature and position of the catalytic base, <i>i</i>.<i>e</i>. Tyr188 that interacts with Glu426 and a monocovalent attachment of the cofactor. In the active site type IIb, nature and position of the catalytic base are conserved (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156892#pone.0156892.g007" target="_blank">Fig 7</a> 1.1 and 1.2) but a bicovalent attachment of the cofactor is to be expected for this active site type. Interestingly the change from a bicovalent to a monocovalent binding mode goes along with the variation of two amino acids of the oxygen reactivity motif. These are the amino acids at position 174 (i.e. cysteine to histidine exchange), responsible for the formation of the covalent bond to C(6) of the isoalloxazine ring and 182 (i.e. histidine to glutamine exchange) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156892#pone.0156892.g007" target="_blank">Fig 7</a>). This is perfectly in line with findings reported by Kopacz <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156892#pone.0156892.ref033" target="_blank">33</a>] on 6-hydroxy-D-nicotine oxidase, a VAO-type flavoprotein with a monocovalent linkage of a histidine to the 8α-position of the isoalloxazine ring. In this enzyme, bicovalent attachment was achieved by introduction of a cysteine and histidine residue corresponding to position 174 and 182, respectively, indicating that the histidine residue plays a crucial role in the formation of the sulfur-carbon bond [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156892#pone.0156892.ref033" target="_blank">33</a>].</p