AuaA, a Membrane‐Bound Farnesyltransferase from Stigmatella aurantiaca, Catalyzes the Prenylation of 2‐Methyl‐4‐hydroxyquinoline in the Biosynthesis of Aurachins

Abstract

Aurachins are quinoline alkaloids isolated from the myxobacterium Stigmatella aurantiaca. They are substituted with an isoprenoid side chain and act as potent inhibitors in the electron transport chain. A biosynthetic gene cluster that contains at least five genes (auaA-auaE) has been identified for aurachin biosynthesis. In this study, auaA, the gene encoding a putative prenyltransferase of 326 amino acids, was cloned and overexpressed in Escherichia coli. Biochemical investigations showed that AuaA catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the presence of farnesyl diphosphate (FPP), thereby resulting in the formation of aurachin D. The hydroxyl group at position C4 of the quinoline ring is essential for an acceptance by AuaA; this was concluded by testing 18 quinoline derivatives or analogues with AuaA and FPP. (1) H NMR and HR-EI-MS analyses of six isolated enzyme products revealed the presence of a farnesyl moiety at position C3 of the quinoline ring. K(M) values of 43 and 270 μM were determined for FPP and 2-methyl-4-hydroxyquinoline, respectively. Like other known membrane-bound prenyltransferases, the reaction catalyzed by AuaA is dependent on the presence of metal ions such as Mg(2+) , Mn(2+) and Co(2+) , although no typical (N/D)DXXD binding motif was found in the sequence.

Mutagenesis and biochemical studies on AuaA confirmed the importance of the two conserved aspartate-rich motifs and suggested difference in the amino acids for substrate binding in membrane-bound prenyltransferases

“…Site-directed mutagenesis One step site-directed mutagenesis protocol (Zheng et al 2004) was used to generate the mutated derivatives of AuaA with plasmid pES19 (Stec et al 2011) as template. , Table S1).…”

“…2). Like other membrane-bound aromatic PTs, AuaA also showed a strict dependency on divalent metal ions (Stec et al 2011). Multiple sequence alignments with several other known membrane-bound aromatic PTs conWrmed the presence of two conserved aspartate-rich motifs (Fig.…”

“…Recently, an additional membrane-bound prenyltransferase, AuaA, was characterized from the myxobacterium S. aurantiaca (Stec et al 2011). AuaA is involved in the biosynthesis of aurachins and catalyzes the transfer of a farnesyl moiety from farnesyl diphosphate to 2-methyl-4-hydroxyquinoline (Fig.…”

A Semipinacol Rearrangement Directed by an Enzymatic System Featuring Dual‐Function FAD‐Dependent Monooxygenase

“…Recent work by Pistorius et al [8] discovered two gene loci containing aurachin biosynthetic genes in addition to the core biosynthetic gene cluster. [18][19][20] The authors speculated that two enzymes encoded by auaG and auaH are responsible for the migration of the farnesyl group from C3 to C4. However, the detailed biosynthetic conversion still remains to be solved, mainly because of the lack of detection of putative intermediates in vivo in auaG and auaH mutants of S. aurantiaca Sg a15.…”

Eine Semipinakol‐Umlagerung – katalysiert durch ein Enzymsystem mit difunktioneller FAD‐abhängiger Monooxygenase

“…Kürzlich zeigten Pistorius et al, [8] dass es zusätzlich zum Genlocus, der für die Biosynthese des Aurachingrundgerüstes kodiert, zwei weitere Genloci gibt, die ebenfalls Gene der Aurachinbiosynthese enthalten. [18][19][20] Die Autoren mutmaßten, dass zwei Enzyme, kodiert als auaG und auaH, für die Wanderung der Farnesylgruppe von C-3 nach C-4 zuständig sind. Allerdings blieb der Biosynthesemechanismus unaufgeklärt.…”