Dihydrofolate reductase(DHFR),a housekeeping enzyme in primary metabolism,has been extensively studied as a model of acid-base catalysis and a clinic drug target.Herein,we investigated the enzymology of a DHFR-like pr...Dihydrofolate reductase(DHFR),a housekeeping enzyme in primary metabolism,has been extensively studied as a model of acid-base catalysis and a clinic drug target.Herein,we investigated the enzymology of a DHFR-like protein SacH in safracin(SAC)biosynthesis,which reductively inactivates hemiaminal pharmacophore-containing biosynthetic intermediates and antibiotics for self-resistance.Furthermore,based on the crystal structure of SacH−NADPH−SAC-A ternary complexes and mutagenesis,we proposed a catalytic mechanism that is distinct from the previously characterized short-chain dehydrogenases/reductases-mediated inactivation of hemiaminal pharmacophore.These findings expand the functions of DHFR family proteins,reveal that the common reaction can be catalyzed by distinct family of enzymes,and imply the possibility for the discovery of novel antibiotics with hemiaminal pharmacophore.展开更多
Summary of main observation and conclusion Collismycins(COLs)are antibiotics characterized by a 2,2'-bipyridine(2,2'-BP)core composed of a trisubstituted ring A and an unmodified ring B.The 2,2'-BP core,wh...Summary of main observation and conclusion Collismycins(COLs)are antibiotics characterized by a 2,2'-bipyridine(2,2'-BP)core composed of a trisubstituted ring A and an unmodified ring B.The 2,2'-BP core,which possesses metal-chelating ability and plays key roles in various biological activities of COLs,is biosynthesized by a nonribosomal peptide synthetase(NRPS)-polyketide synthase(PKS)hybrid machinery.The starter module of the NRPS-PKS hybrid machinery consists of a type II peptidyl carrier protein(PCP)ColA1a and an adenylation protein ColA1b.We here report the functional characterization of ColAia and ColAlb in vitro,confirming their functions in selection and loading of picolinic acid(PA),instead of normal amino acid substrates,as the origin of ring B in COLs.The 2.1 A crystal structure of ColAia was solved,revealing structural features including the additional helices ala,alb and missing helix a3,which may reflect unique interactions of ColAia with other NRPS-PKS proteins/domains or substrate.Primary and tertiary structural comparison of ColAia with other PCPs revealed the structural basis for their typical a-helical bundle,providing a better understanding of the structural flexibility of PCPs.These results facilitate the starter module engineering for the generation of COL derivatives with ring B modifications in the future.展开更多
基金supported by the National Natural Science Foundation of China(Nos.31930002,21621002,21877002,81991525,82273829 and 22077007)the key project at central government level:the ability establishment of sustainable use for valuable Chinese medicine resources(2060302-2201-17).
文摘Dihydrofolate reductase(DHFR),a housekeeping enzyme in primary metabolism,has been extensively studied as a model of acid-base catalysis and a clinic drug target.Herein,we investigated the enzymology of a DHFR-like protein SacH in safracin(SAC)biosynthesis,which reductively inactivates hemiaminal pharmacophore-containing biosynthetic intermediates and antibiotics for self-resistance.Furthermore,based on the crystal structure of SacH−NADPH−SAC-A ternary complexes and mutagenesis,we proposed a catalytic mechanism that is distinct from the previously characterized short-chain dehydrogenases/reductases-mediated inactivation of hemiaminal pharmacophore.These findings expand the functions of DHFR family proteins,reveal that the common reaction can be catalyzed by distinct family of enzymes,and imply the possibility for the discovery of novel antibiotics with hemiaminal pharmacophore.
基金National Natural Science Foundation of China(Grant No.81741148,81573326,81673332,21877002)COMRA Project of China(Grant No.DY135-B2-08)China Postdoctoral Science Foundation(Grant No.2018M641123)
基金This research was supported in part by the National Natural Science Foundation of China(Nos.81673332,81573326,21877002 and 81991525)the China Postdoctoral Science Foundation(Nos.2019M660362 and 2018M641123).
文摘Summary of main observation and conclusion Collismycins(COLs)are antibiotics characterized by a 2,2'-bipyridine(2,2'-BP)core composed of a trisubstituted ring A and an unmodified ring B.The 2,2'-BP core,which possesses metal-chelating ability and plays key roles in various biological activities of COLs,is biosynthesized by a nonribosomal peptide synthetase(NRPS)-polyketide synthase(PKS)hybrid machinery.The starter module of the NRPS-PKS hybrid machinery consists of a type II peptidyl carrier protein(PCP)ColA1a and an adenylation protein ColA1b.We here report the functional characterization of ColAia and ColAlb in vitro,confirming their functions in selection and loading of picolinic acid(PA),instead of normal amino acid substrates,as the origin of ring B in COLs.The 2.1 A crystal structure of ColAia was solved,revealing structural features including the additional helices ala,alb and missing helix a3,which may reflect unique interactions of ColAia with other NRPS-PKS proteins/domains or substrate.Primary and tertiary structural comparison of ColAia with other PCPs revealed the structural basis for their typical a-helical bundle,providing a better understanding of the structural flexibility of PCPs.These results facilitate the starter module engineering for the generation of COL derivatives with ring B modifications in the future.
