Unveiling Of The Novel Enzymatic Reactions Involved In The Assembly Of Cabazole "A" Ring And The Attached Alkyl Side Chain Using Neocarazostatin A As The Model

For several decades,alkaloids have been one of the hottest topics in the research field of natural products due to their excellent pharmaceutical activities as well as their rich structural diversities.We choose Neocarazostatin A as the target compound in this study because it possesses a great potential to be developed into a novel antioxidant prodrug based on its strong inhibitory effect on free radicals induced brain lipid peroxidation.The mechanisms underlying the biosynthesis of the carbazole"A" ring of Neocarazostatin A and the C4 alkyl side chain attached on "A" ring still remains elusive.Bioinformatics analysis of Neocarazostatin A biosynthetic gene cluster has suggested that five enzymes may participate in the above process,including NzsE/F/H/I/J.In this study,we have investigated the reactions catalyzed by the acetolactate synthase NzsH,the acyl carrier protein NzsE,and the ?-ketoacyl-acyl carrier protein synthase III NzsF.Our results indicate that NzsH is a thiamine diphosphate(ThDP)dependent enzyme which catalyzes an acyloin coupling reaction between indole-3-pyruvate and pyruvate to initiate the assembly of the carbazole "A" ring.On the other hand,NzsF catalyzes a decarboxylative condensation between an acetyl-CoA and the NzsE bound malonyl thioester to generate acetoacetyl-NzsE,which is further reduced by MA37-FabG,the NADPH-dependent reductase from the fatty acid biosynthesis pathway,to produce the precursor for the C4 alkyl side chain.In summary,this study successfully reconstitutes the major part of the biosynthetic pathway of the carbazole "A" ring in vitro,which not only enriches our knowledges on ThDP-dependent enzyme family,but also strengthens our understanding of the crosstalk between the primary and secondary metabolisms in Streptomyces.Our works therefore pave the way for future engineering of NzsE/F for producing novel neocarazostatin analogues in the host strain,as well as holds potential for unveiling the whole enigmatic bacterial carbazole "A" ring biosynthesis.