As the most well-studied group of signalling molecules in Streptomyces, the biosynthesis of GBLs has attracted a great deal of interests since the discovery of the first γ-butyrolactone molecule A-factor. Investigation into A-factor biosynthesis was first conducted by Horinouchi and co-workers as early as 1984. A-factor production was restored by insertion of the afsA gene into an A-factor-deficient S. griseus mutant, indicating that AfsA may be an important enzyme required for A-factor biosynthesis.63-
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Prior to identification of the specific enzymes responsible for production of GBLs, the biosynthesis of VB-A 33 was investigated by incorporation experiments using stable isotope-labelled precursors in Streptomyces antibioticus.66 The feeding experiments
revealed that VB-A is formed from two molecules of acetate and one molecule of both glycerol and isovalerate (Figure 1.7). It was proposed that β-keto acyl CoA is the key precursor, which couples to a C3 unit that is oxidised from the glycerol molecule.
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Figure 1.7. Incorporation results using 13C-labelled glycerol, acetate and isovalerate for
investigation of VB-A 33 biosynthesis by Sakuda and co-workers.66
The biosynthesis of GBLs didn’t become much clearer until in vitro reconstitution of AfsA by Kato and co-workers in 2007.67 AfsA has been demonstrated to be the key enzyme required for A-factor biosynthesis, catalysing β-ketoacyl transfer from an acyl carrier protein (ACP)-bound 8-methyl-3-oxononanoyl thioester 51 to the hydroxyl group of dihydroxyacetone phosphate (DHAP) 50 to form an 8-methyl-3-oxononanoyl-DHAP ester intermediate 52 (Scheme 1.1). After formation of the phosphorylated ester intermediate 52, three further reactions are required for A-factor biosynthesis, including an intramolecular aldol reaction that leads to the butenolide structure 55, a reduction step for the C2-C3 double bond in the butenolide intermediate and a dephosphorylation step to give the final A-factor 28. It was also proposed that the three subsequent steps may occur in a different order, i.e. dephosphorylation of the ester intermediate 52 is followed by the aldol condensation and final reduction of the butenolide 54 to A-factor (Scheme 1.1).
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Scheme 1.1. Biosynthetic route to A-factor as proposed by Kato and co-workers.
A range of C3 compounds were first tested with His-AfsA, leading to identification of DHAP 50 as the C3 substrate for the AfsA-catalysed reaction. Then the enzymatic assay was undertaken using recombinant His-AfsA, 32P-labelled DHAP and the synthetic 8- methyl-3-oxononanoyl-N-acetylcysteamine (NAC), which mimics the corresponding β- ketoacyl-ACP. The reaction resulted in a product with radioactivity on radio-thin layer chromatography (TLC), which was demonstrated to be the ester intermediate, 8-methyl- 3-oxononanoyl-DHAP ester 52, by comparing to its synthetic standard. The butenolide is then formed by a nonenzymatically catalysed intramolecular aldol reaction of the ester intermediate 52.
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BprA (butenolide phosphate reductase), an NADPH-dependent reductase that is encoded by a gene directly downstream of afsA, was confirmed in vitro to be responsible for reduction of the butenolide 55 to form the butanolide 56. However, it was shown that the phosphatases and reductases commonly present in bacteria are able to catalyse similar reactions to afford A-factor. For example, introduction of afsA into E. coli caused the host to produce A-factor analogues, with straight side chains, due to the differences in fatty acid metabolism between Streptomyces species and E. coli. Streptomyces species can produce branched fatty acids from starter units such as isobutyryl-CoA and methylbutyryl-CoA from amino acid degradation, whereas E. coli predominantly uses acetyl CoAand thus does not produce branched fatty acids.68-69
An AfsA homologue, ScbA in S. coelicolor, has been proposed for the biosynthesis of SCBs.70-71 Takano and co-workers conducted in silico analysis of the AfsA family of proteins, including ScbA, and showed that they have similarity to the fatty acid synthesis enzymes FabA and FabZ. Meanwhile, mutation of two predicted active sites in ScbA led to abolishment of the production of SCBs in S. coelicolor. Other AfsA homologues have also been found in corresponding γ-butyrolactone producers, e.g. BarX in S. virginiae
and FarX in S. lavendulae.72-73 It is reasonable to assume that these homologues have similar activity as AfsA in γ-butyrolactone biosynthesis. It was also suggested by Kato and co-workers that a BprA orthologue, ScbB, which has 76% identity in amino acid sequence to BprA, catalyses the reduction of C2-C3 double bond to form the butanolide phosphate 56 in SCB biosynthesis.67
Both SCBs in S. coelicolor A3(2) and VBs in S. virginiae contain a hydroxyl group at C-6 instead of the keto group in A-factor, indicating the presence of additional reductases for this reduction. Due to the absence of such reductase in S. griseus, C-6 of A-factor remains as a keto group. One such enzyme, BarS1 in S. virginiae, has been characterised. BarS1 was isolated as an NADPH-dependent reductase that reduces the 6-oxo group of the penultimate intermediate in the VB biosynthesis.74 A BarS1 orthologue, SCO6264,
has been proposed to be involved in SCB biosynthesis for a similar reduction reaction in
S. coelicolor.70 More investigations are needed to experimentally determine the specific
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