Structural Basis for β-Carboline Alkaloid Production by the Microbial Homodimeric Enzyme McbB

Abstract

The β-carboline (βC) alkaloids occur throughout nature and exhibit diverse biological activities. In contrast to βC alkaloid synthesis in plants, the biosynthesis in microorganisms remains poorly understood. The recently reported McbB from Marinactinospora thermotolerans is a novel enzyme proposed to catalyze the Pictet-Spengler (PS) reaction of L-tryptophan and oxaloacetaldehyde to produce the βC scaffold of marinacarbolines. In this study, we solved the crystal structure of McbB complexed with L-tryptophan at 2.48 Å resolution, which revealed the novel protein folding of McbB and the totally different structure from those of other PS condensation catalyzing enzymes, such as strictosidine synthase and norcoclaurine synthase from plants. Structural analysis and site-directed mutagenesis confirmed that the previously proposed catalytic Glu97 at the active-site center functions as an acid and base catalyst. Remarkably, the structure-based mutants R72A and H87A, with expanded active-site cavities, newly accepted bulky phenylglyoxal as the aldehyde substrate, to produce 1-benzoyl-3-carboxy-β-carboline.

Increasing the Diversity of Known Pictet-Spenglerases

“…The current in vitro studies by Mori et al (2015) confirmed that purified McbB converts L-tryptophan and oxaloacetaldehyde into the b-carbolines 1 (major product) and 2 (minor product). They also demonstrated that a variety of other simpler aldehydes, such as acetaldehyde and isobutyraldehyde, served as alternate substrates, but the non-enzymatic oxidation/ decarboxylation steps that aromatize the newly formed ring did not take place.…”

“…The C domain of the nonribosomal peptide synthetase SfmC has also been reported to catalyze iterative Pictet-Spengler reactions in the biosynthesis of saframycin A, although structural information on this enzyme is absent (Koketsu et al, 2010). In this issue, Mori et al (2015) report the structural elucidation of a new type of Pictet-Spenglerase, McbB, which is involved in the biosynthesis of b-carboline alkaloids (Mori et al, 2015). While the amine-containing substrate in this reaction is provided by L-tryptophan, the structure of McbB is unrelated to that of either strictosidine synthase or norcoclaurine synthase, and thus it represents a novel evolutionary answer to the answer of Pictet-Spenglerase catalysis.…”

Enzyme catalysed Pictet-Spengler formation of chiral 1,1’-disubstituted- and spiro-tetrahydroisoquinolines

“…Pictet–Spenglerases have also been identified in bacterial secondary metabolism151617. The plant enzymes have a central role in engineered microbial systems for the production of alkaloids1819202122.…”

Die Enzymologie organischer Umwandlungen: Namensreaktionen in biologischen Systemen

“…McbB aus Marinactinospora thermotolerans katalysiert die Kondensation von l ‐Tryptophan ( 70 ) mit Oxaloacetaldehyd ( 71 ) zu β‐Carbolin ( 72 ). Obwohl McbB eine andere Proteinfaltung besitzt als Strictosidin‐Synthase, wurde Glu97 anhand von Untersuchungen zur ortsspezifischen Mutagenese von McbB als katalytisch wirksamer Rest identifiziert . Dieser strukturelle Hinweis spricht dafür, dass die Katalysen von Kondensationsreaktionen durch McbB und Strictosidin‐Synthase wahrscheinlich nach ähnlichen Mechanismen ablaufen.…”