Bifurcate evolution of quinone synthetases in basidiomycetes.

BACKGROUND: The terphenylquinones represent an ecologically remarkable class of basidiomycete natural products as they serve as central precursors of pigments and compounds that impact on microbial consortia by modulating bacterial biofilms and motility. This study addressed the phylogenetic origin of the quinone synthetases that assemble the key terphenylquinones polyporic acid and atromentin. RESULTS: The activity of the Hapalopilus rutilans synthetases HapA1, HapA2 and of Psilocybe cubensis PpaA1 were reconstituted in Aspergilli. Liquid chromatography and mass spectrometry of the culture extracts identified all three enzymes as polyporic acid synthetases. PpaA1 is unique in that it features a C-terminal, yet catalytically inactive dioxygenase domain. Combined with bioinformatics to reconstruct the phylogeny, our results demonstrate that basidiomycete polyporic acid and atromentin synthetases evolved independently, although they share an identical catalytic mechanism and release structurally very closely related products. A targeted amino acid replacement in the substrate binding pocket of the adenylation domains resulted in bifunctional synthetases producing both polyporic acid and atromentin. CONCLUSIONS: Our results imply that quinone synthetases evolved twice independently in basidiomycetes, depending on the aromatic α-keto acid substrate. Furthermore, key amino acid residues for substrate specificity were identified and changed which led to a relaxed substrate profile. Therefore, our work lays the foundation for future targeted enzyme engineering.

Evaluation of γ-carboline-phenothiazine conjugates as simultaneous NMDA receptor blockers and cholinesterase inhibitors.

Alzheimer's disease (AD) is the most common form of dementia. It is associated with the impairment of memory and other cognitive functions that are mainly caused by progressive defects in cholinergic and glutamatergic signaling in the central nervous system. Inhibitors of acetylcholinesterase (AChE) and ionotropic glutamate receptors of the N-methyl-d-aspartate (NMDA) receptor family are currently approved as AD therapeutics. We previously showed using a cell-based assay of NMDA receptor-mediated glutamate-induced excitotoxicity that bis-γ-carbolinium conjugates are useful NMDA receptor blockers. However, these compounds also act as subnanomolar AChE inhibitors, which may cause serious anticholinergic side effects when applied in vivo. Here, we evaluated new structures containing γ-carbolines linked to phenothiazine via a propionyl spacer. These compounds were superior to the previously characterized bis-γ-carbolinium conjugates because they blocked NMDA receptors without requiring a quaternary pyridine N-atom and inhibited AChE with moderate IC50 values of 0.54-5.3 µM. In addition, these new compounds displayed considerable selectivity for the inhibition of butyrylcholinesterase (BChE; IC50 = 0.008-0.041 µM), which may be favorable for AD treatment. Inhibitory activities towards the NMDA receptors and AChE were in the same micromolar range, which may be beneficial for equal dosing against multiple targets in AD patients.