Tropane alkaloids biosynthesis involves an unusual type III polyketide synthase and non-enzymatic condensation

Abstract

The skeleton of tropane alkaloids is derived from ornithine-derived N -methylpyrrolinium and two malonyl-CoA units. The enzymatic mechanism that connects N -methylpyrrolinium and malonyl-CoA units remains unknown. Here, we report the characterization of three pyrrolidine ketide synthases (PYKS), Aa PYKS, Ds PYKS, and Ab PYKS, from three different hyoscyamine- and scopolamine-producing plants. By examining the crystal structure and biochemical activity of Aa PYKS, we show that the reaction mechanism involves PYKS-mediated malonyl-CoA condensation to generate a 3-oxo-glutaric acid intermediate that can undergo non-enzymatic Mannich-like condensation with N -methylpyrrolinium to yield the racemic 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This study therefore provides a long sought-after biosynthetic mechanism to explain condensation between N -methylpyrrolinium and acetate units and, more importantly, identifies an unusual plant type III polyketide synthase that can only catalyze one round of malonyl-CoA condensation.

The scaffold-forming steps of plant alkaloid biosynthesis

“…Recently, the enzymes that are required to form the characteristic seven-membered tropane ring have been described. [152][153][154] Pyrrolidine ketide synthase (PYKS), a type III polyketide synthase, catalyses the formation of 3-oxoglutaric acid from two malonyl-CoA units. PYKS has an active site with the typical type-III PKS catalytic triad for substrate loading and chain extension (Cys166, His305 and Asn338), but has additional residues (Arg-134 and Ser-340) which limit the enzyme to a single extension step by interacting with the substrate carboxylate moiety.…”

“…PYKS has an active site with the typical type-III PKS catalytic triad for substrate loading and chain extension (Cys166, His305 and Asn338), but has additional residues (Arg-134 and Ser-340) which limit the enzyme to a single extension step by interacting with the substrate carboxylate moiety. 153 When incubated with PYKS, N-methylpyrollinium and malonyl-CoA form 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid, a key intermediate. 152,153 PYKS forms 3-oxoglutaric acid from malonyl-CoA and releases it into the solvent where it spontaneously reacts with N-methylpyrollinium through a decarboxylative Mannich condensation.…”

“…153 When incubated with PYKS, N-methylpyrollinium and malonyl-CoA form 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid, a key intermediate. 152,153 PYKS forms 3-oxoglutaric acid from malonyl-CoA and releases it into the solvent where it spontaneously reacts with N-methylpyrollinium through a decarboxylative Mannich condensation. 153 PYKS does not utilise N-methylpyrollinium as a starter unit, nor does the enzyme catalyse the condensation reaction.…”

“…152,153 PYKS forms 3-oxoglutaric acid from malonyl-CoA and releases it into the solvent where it spontaneously reacts with N-methylpyrollinium through a decarboxylative Mannich condensation. 153 PYKS does not utilise N-methylpyrollinium as a starter unit, nor does the enzyme catalyse the condensation reaction. This observation is supported by the racemic nature of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid, and by the presence of the side products hygrine and cuscohygrine, which may be formed through decarboxylation or a second decarboxylative Mannich condensation respectively.…”

Synthesis and Antimalarial Activity of 1,4-Disubstituted Piperidine Derivatives

“…The 4-arylaminopiperidine is a structural moiety found in many alkaloids [4][5][6][7][8][9][10][11] and pharmaceutical products such as fentanyl and structurally-related analgesic opioids or H1-antihistamines agents such as bamipine [12][13][14][15][16][17] and neurokinin 1 (NK1) receptor antagonists [18][19][20]. Studies have shown that compounds with piperidine rings [4,8,[21][22][23][24][25][26] have good selectivity and activity for the P. falciparum strain.…”