ABREVIATIONS: ACP – acyl carrier protein, SAM – S-adenosyl-methionine, CoA – coenzyme A
INTRODUCTION
For enabling the outside-of-cell activity of the recombinant LuxI enzyme which will serve as the signal transducing element in our system we needed to provide the substrates for it: first S-adenosyl-methionine (SAM) which also serves as a universal donor in methylation reactions and is easy to get from the store and second acylated-acyl carrier protein (in our case hexanoyl-ACP) which is not readily available commercially but IS possible to synthesize by more available strategies. The synthesis and detection of this compound is the topic here.
SYNTHESIS:
There are two major quorum signaling molecules produced by the V. fischeri LuxI enzyme in almost equimolar concentrations: N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone 1 which differ by just a keto group at the third carbon of the acyl chain, however the second compound gives us the great advantage to synthesize it using commercially available products which is why we adopted this approach.
Normally the in-vivo synthesis of the acyl-ACP substrate for the production of signaling molecules in bacterial quorum sensing is carried out by enzymes like acyl-ACP synthetases which can transfer fatty acid groups onto the functional ACPs (already containing the phosphopantetheine prosthetic group).
We, however, tried an entirely different synthesis approach by using a different enzyme, namely ACP synthase (4-phosphopantetheinyl-transferase) which is involved in a more general reaction that spans all the domains of life and represents the addition of a prosthetic group to an ACP protein either from the fatty acid or polyketide synthesis pathways in order to make it functional. The prosthetic group in this case is a phosphopantetheine which is taken directly from CoA and added to the apo-protein variant of ACP generating adenosine 3',5'-bisphosphate and holo-ACP (reaction depicted below).
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