Studies On The Function Of Condensing Enzyme RedH And SDR Family Oxidoreductase HbnC In Hybrubin Biosynthesis

Hybrubins including hybrubins A,B,and C,is a class of “non-natural” red alkaloids with the same 4'-methoxy-2,2'-bipyrrole-5'-methine moiety as prodiginines and a new C-ring derived from tetramic acids(2,4-pyrrolidinedione).Due to the novel chimera structure that a tetramic acid ring joins to a 2,2'-bipyrrole motif and the unique biological activities of hybrubins,which motivated this thesis to study their biosynthetic mechanism.In the early stage of the research,our group found that hybrubins were generated by two convergent biosynthetic pathways including the truncated genomic red cluster and the BAC encoded hbn genes.Considering previously reported the relaxed substrate-specificity of enzyme,it was hypothesized that 4'-methoxy-2,2'-bipyrrole-5'-carbaldehyde(MBC)and(Z)-5-ethylidenetetramic acid(ETA)may be condensed to produce hybrubins A and B under the catalysis of RedH and hybrubins A and B were further oxidized to produce hybrubin C.However,the C5'-carbaldehydederived methine bridge in hybrubin is attached to the C-3'' position of C-ring(tetramic acid),while the C5' methine bridge in undecylprodigiosin is connected the C-ring(monopyrrole)at the C5'' position.On the other hand,tetramic acids can spontaneously react with electrophiles such as aldehydes.Therefore,it is questionable that both whether RedH can catalyze a novel condensation reaction and whether the condensation of MBC with tetramic acid requires enzyme catalysis.To confirm whether undecylprodigiosin synthetase gene redH correlate with the biosynthesis of hybrubin,genetic experiments such as knockout and complementation were first carried out.The results showed that the ?redH mutant completely interrupted the biosynthesis of hybrubins A and B,while the yield of hybrubin C was not abolished but rather increased to about twice the level of the wild-type,indicating that RedH plays a role in the biosynthesis of hybrubins A and B but not in hybrubin C and implying that the biosynthesis of hybrubin C may be on a branch pathway.The complementation strain restored the production of hybrubins A and B,which validated that the activity of RedH is essential for the biosynthesis of hybrubins A and B.In vitro biochemical assays showed that MBC and ETA could condense to form hybrubin B under the catalysis of RedH.These results demonstrated that RedH was responsible for the critical step of MBC-ETA condensation in hybrubin biosynthesis.It was further found that the catalysis of RedH was ATP-dependent in the reaction.In this thesis,the homologue of RedH in Serratia prodigiosin synthase PigC was also studyed.In vitro biochemical experiments showed that PigC has a similar function as RedH,and can also catalyze the intramolecular condensation between MBC and ETA to produce hybrubin B.To investigate the substrate scope of RedH and PigC,a series of chemically synthesized ETA analogues were selected as C-ring substitutes.The results showed that when the C-ring at the C-5 position was unsubstituted,or the substituent was a alkyl or a alkenyl group such as methyl,propyl,1-methylethyl or propylidene,or the ETA was replaced with a tetronic acid,each of them can be condensed with MBC under the catalysis of RedH/PigC to produce hybrubin derivatives.However,when the 4-keto group of the C-ring was reduced to 4-hydroxyl,or larger rings were used as substrates,the reaction did not occurred.These results suggested that RedH and PigC have good adaptability to the C-ring substrates.Both enzymes could catalyzed the condensation reaction between MBC and various tetramic acids or tetronic acid,in addition to the natural substrates 2-UP and MAP.New hybrubin derivatives 5a-5f produced in above reaction were identified by high-resolution electrospray ionization mass spectrometry(HR-ESI-MS)and tandem MS/MS.Bioinformatics analysis revealed that RedH belongs to the pyruvate-phosphate dikinase(PPDK)superfamily.Three key amino acid residues of RedH in S.lividans,including Glu-307 and Arg-321 of the ATP-binding domain and His-869 of the phosphoryl transfer domain,were indentified by multiple sequence alignments with conserved domains of classical PPDK,phosphoenolpyruvate synthase(PEPS)and prodiginine synthetases such as PigC and Hap C.Subsequently,the predicted key amino acid residues were mutated to alanine by site-directed mutagenesis.In vitro characterization of the RedH variants showed that the E307 A and H869 A mutants completely lost their catalytic activities in the MBC-ETA condendation,while the activity of the R321 A mutant drastically reduced to approximately 10% of the level of wild-type.These results indicated that both the ATP-binding and phosphoryltransferring functions of RedH were essential for the MBC-ETA condensation,implying that the RedH-catalyzed reaction proceeds via a putative phosphorylated MBC intermediate,analogous to the proposed mechanism of PigC in prodigiosin biosynthesis.To detect putative phosphorylated intermediate and to verify the hypothesis about the condensation mechanism,the MBC-TAs condensation were separated into two half reactions.In the first half of the reaction,the first substrate MBC and co-substrate ATP were incubated with RedH or PigC.The reaction mixture was subjected to methanol extraction after boiling,and the extract was dectected by HR-ESI-MS and MS/MS analysis.The results revealed and confirmed the presence of the putative phosphorylated MBC(Pi-MBC)in the RedH/PigC-catalyzed reaction.In the second half of the reaction,ETA or tetramic acid 8a was incubated with Pi-MBC,which produced hybrubin B or its derivative 5a comparable to the one-pot reaction.These results suggested that RedH-or PigC-catalyzed MBC-TAs condensation was actually performed by a two-step style: first,the enzyme catalyze the formation of the intermediate Pi-MBC;then Pi-MBC spontaneously condenses with tetramic acids to generate the final product.To elucidate the biosynthetic pathway of hybrubin C,the single-gene knockout and corresponding complementation on hbn gene cluster were performed.The results showed that the production of hybrubin C was significantly decreased in the ?hbn C mutant while the yield of hybrubins A and B were increased.Moreover,the complementation of hbn C restored the hybrubin C production.These results suggested that the hbn C was required for the biosynthesis of hybrubin C and HbnC was presumed to be responsible for the conversion of(Z)-5-ethylidenetetramic acid to 5-ketotetramic acid.Bioinformatics analysis revealed that HbnC belongs to the oxidoreductase of the short-chain dehydrogenase/reductase(SDR)family.In the end,a preliminary exploration was conducted to the function of HbnC in vitro.In summary,first,this thesis demonstrated that RedH,from the host S.lividans undecylprodigiosin biosynthetic pathway,was responsible for the biosynthesis of hybrubins A and B,and in vitro biochemical reactions confirmed that RedH and homologue PigC can catalyze the intermolecular condensation of MBC with(Z)-5-ethylidenetetramic acid to generate hybrubin B.Second,it was observed that RedH/PigC can catalyze the condensation between MBC and diverse tetramic acids or a tetronic acid to yiled hybrubin derivatives,exhibiting excellent tolerance to substates on the C-ring.Last,further studies demonstrated that RedH and PigC activated MBC via phosphorylation of the aldehyde group to form an intermediate Pi-MBC and then the condensation of Pi-MBC with tetramic acids or tetronic acid occurs in a nonenzymatic style to generate final product,which explained the substrate promiscuity of RedH/PigC in one-pot MBC-TAs condensation and greatly enriched the members of the hybrubin family and provided a new way for producing novel hybrubin derivatives.In addition,this thesis revised the hybrubin biosynthetic pathway and laid a genetic basis for elucidating the biosynthesis of hybrubin C.