Molecular Mechanism Of Substrate Specificity For Acyl Units Of Acyltransferase In Tacrolimus Biosynthetic Polyketide Synthase

Tacrolimus(FK506),a secondary metabolite of Streptomyces tsukubaensis,is well known as an immunosuppressive agent for organ transplantation,which is 50 times more effective than cyclosporine.It inhibit the activation of the transcription factors NF-ATc which is Ca2+-CaN-sensitive subunits of nuclear factor of activated T cell transcription complexes,and the transcription of the tinterleukin-2 gene by regulating calcineurin.FK506 backbone is catalyzed by the type Ⅰ polyketide synthase(PKS)which is an important pathway for the biosynthesis of microbial natural products and contains multifunctional enzyme,for example acyltransferase(AT)domain.AT domain of PKS is critical for selection of acyl units to acyl carrier protein(ACP)domain by self-acylation and trans-acylation reactions to produce a variety of polyketide.Therefore,the substrate specificity of AT domains is one of the key factors on determining the diversity of polyketide backbones.However,there is little known regarding the substrate specificity of AT.An industrial strain Streptomyces tsukubaensis L19 was as material in our experiments and its whole genomic DNA was sequenced to identify the FK506 biosynthetic gene cluster.The protein sequences corresponding to the module and domain of FK506 PKSs in S.tsukubaensis L19 are identical with those corresponding to the module and domain of FK506 PKSs in Streptomyces sp.KCTC 11604BP(GenBank accession number HM116537).Here,we study the substrates specificity of AT4FkbB and AT8FkbA in S.tsukubaensis L19.The main results are as follows:1.S.tsukubaensis L19 produces both FK506 and its analogue FK520 as secondary metabolites and the strcutres of those two compounds are that the side chain of FK506 is allyl unit while FK520 is ethyl unit.We selected AT4FkbB as a model to study the substrate specificity and the key residues for selection of allymalonyl(AM)unit to interpret the production of FK506 and FK520 and reduce the field of FK520 by AT site-directed mutagenesis.In the biochemical experiments,we found that AT4FkbB recognizes both ACPTcsA and CoA as acyl donors for transfer of a unique AM unit to ACP4FkbB,resulting in FK506 production.In addition,AT4FkbB uses CoA as an acyl donor to transfer an unusual ethylmalonyl(EhM)unit to ACP4FkbB,resulting in FK520 production and transfers AM unit to non-native ACP acceptors.And AT4FkbB controls the transfer of AM and EM not malonyl(M)or methylmalonyl(MM)in trans-acylation reactions.What’s more,AT4FkbB transfers both AM and EM units to diverse ACPs.Mutations of five residues in AT4FkbB(Q119A,L185I-V186D-V187T and F203L)caused decreased efficiency of AM transfer,but a higher ratio of EM transfer,supposedly due to less nucleophilic attacks between Ser599 in the active site of AT4Fkbs and the carbonyl carbon in the AM unit.Furthermore,reverse mutations of these five residues in EM-specific ATs to the corresponding residues of AT4FkbB increased its binding affinity to AM-CoA.Among these residues,Val187 of AT4pFkbB mainly contributed to AM recognition,and the ratio of FK506 and FK520 in V187K was 7.7:1 compared to 6.1:1 in wild type(WT),suggesting that V187K can produce high FK506 and less FK520.2.The methoxylmalonyl(MeO)unit which is biosynthesed by subgene cluster fkbG～fkbL of FK506,is another important acyl unit of FK506 backbone.In this paper,we study the substrate specificity of AT7FkbA,AT8FkbA,ATIOFkbA and the key residues for selection of MeO unit to explain the molecular mechanism of MeO unit involved in the biosynthesis of type I polyketide backbone and produce new FK506 derivative by AT site-directed mutagenesis.In biochemical assays,we found that AT7FkbA and AT8FkbA recognizes both ACPFkbJ and CoA as acyl donors for transfer of a unusual AM unit to ACP7FkbA and ACP8FkbA respectively to introduce MeO unit into the biosynthesis of the FK506 backbone.What’s more,AT8FkbA controls the transfer of MeO not M,MM,EM or AM in self-acylation reactions.Furthermore,Leu75,Gly169,His160 and His161 of AT8FkbA play the important roles in transfer of MeO unit by the amino acids alignment of MeO-specific ATs,the molecular dynamics simulations(MDs)of MeO-[KS8][AT8]FkbA and docking of[KS8][AT8]-ACP8FkbA,the biochemical assays of AT8FkbA point mutants.Because of the similar amino acid sequences between AT8FkbA and AT10FkMA,the relevant sites on AT10FkbA were mutated to Leu75,Gly 169,His 160 and His161 of AT8FkbA.The results showed that AT 10FkbA mutants could transfer MeO unit not AM,EM and MM to ACP10FkbA in traps-acylation reactions,resulting the production of FK506 derivative,suggesting that the above 4 residues control the transfer of MeO unit.The study about the molecular mechanism of substrate specificity of AT domain reveals the mystery of the diversity of polyketides biosynthesis and it is an important theoretical basis for the synthetic biology of polyketide drugs.Based on the above mechanism,we can introduce the desired acyl units into the backbone by key point mutations of AT domain for further producing the high-yield and target polyketides or the novel artificial natural products.Therefore,this study has a wide range of application prospects.