Probing the acyl carrier protein–Enzyme interactions within terminal alkyne biosynthetic machinery

Abstract

The alkyne functionality has attracted much interest due to its diverse chemical and biological applications. We recently elucidated an acyl carrier protein (ACP)-dependent alkyne biosynthetic pathway, however, little is known about ACP interactions with the alkyne biosynthetic enzymes, an acyl-ACP ligase (JamA) and a membrane-bound bi-functional desaturase/acetylenase (JamB). Here, we showed that JamB has a more stringent interaction with ACP than JamA. In addition, site directed mutagenesis of a non-cognate ACP significantly improved its compatibility with JamB, suggesting a possible electrostatic interaction at the ACP-JamB interface. Finally, error-prone PCR and screening of a second non-cognate ACP identified hot spots on the ACP that are important for interacting with JamB and yielded mutants which were better recognized by JamB. Our data thus not only provide insights into the ACP interactions in alkyne biosynthesis, but it also potentially aids in future combinatorial biosynthesis of alkyne-tagged metabolites for chemical and biological applications.

Structures and Biosynthesis of Caryoynencins, Unstable Bacterial Polyynes from Pseudomonas protegens Recombinant Expressing the cayG Gene

“…The proposed biosynthetic pathway of protegenin A ( 6 ) and caryoynencins in Cab57- cayG is shown in Figure D. First, ProA (fatty acyl-AMP ligase) activates the precursor (stearic acid), loading it into ProD (ACP) and yielding a stearyl-ACP conjugate. , Thereafter, ProB (desaturase/acetylenase), ProC (desaturase/acetylenase), and ProE (desaturase domain) successively act on stearyl-ACP to construct the ene–tetrayne moiety. , ProF may be involved in electron transfer during the ene–tetrayne formation . The thioesterase activity of ProE causes the fatty acid part to be cleaved from the ACP complex, thereby yielding protegenin A ( 6 ). ,, From the amino acid sequence, ProG (α/β-hydrolase) was expected to play a role in assisting the thioesterase activity of ProE.…”

“…First, ProA (fatty acyl-AMP ligase) activates the precursor (stearic acid), loading it into ProD (ACP) and yielding a stearyl-ACP conjugate. 25,26 Thereafter, ProB (desaturase/acetylenase), ProC (desaturase/acetylenase), and ProE (desaturase domain) successively act on stearyl-ACP to construct the ene−tetrayne moiety. 23,24 ProF may be involved in electron transfer during the ene−tetrayne formation.…”

Untargeted Identification of Alkyne-Containing Natural Products Using Ruthenium-Catalyzed Azide Alkyne Cycloaddition Reactions Coupled to LC-MS/MS

“…Coupled with the fact that only a handful of polyyne natural product biosynthetic gene clusters (BGCs) have been identified to date makes it difficult to identify putative polyyne-producing organisms using antiSMASH . Previous studies have been conducted on the biosynthesis of bacterial acetylenic natural products. ,, These studies have determined that a desaturase is most likely responsible for the formation of the alkyne bond. Along with a desaturase, an acyl carrier protein and an AMP-dependent ligase are thought to play important roles in the biosynthesis of the alkyne moiety .…”

“…22 Previous studies have been conducted on the biosynthesis of bacterial acetylenic natural products. 4,23,24 These studies have determined that a desaturase is most likely responsible for the formation of the alkyne bond. Along with a desaturase, an acyl carrier protein and an AMP-dependent ligase are thought to play important roles in the biosynthesis of the alkyne moiety.…”

Biosynthesis of alkyne-containing natural products

“… 65,66 Zhang's group further demonstrated that desaturase/acetylenase (JamB) has a more stringent interaction with ACP than the acyl-ACP ligase (JamA) by both in vitro and in vivo assays, which provided new insights into the role of ACP in alkyne biosynthesis. 67 …”