| Polycyclic tetramate macrolactam(PoTeM)is a class of macrolactams with a tetramate acid unit and a polycyclic system,which has novel chemical structures and extensive biological activities.According to the polycyclic system,PeToMs can be divided into 5-5 bicyclic,5-5-6 tricyclic and 5-6-5 tricyclic compound and other structural subtypes.Biosynthesis studies have shown that the alcohol dehydrogenase OX4 is responsible for the inner 6-membered ring formation of 5-5-6 tricyclic HSAF and the alcohol dehydrogenase CftD catalyzed the inner 5membered ring formation of 5-6-5 tricyclic ikarugamycin.So far,none of the crystal structures of PoTeMs-related alcohol dehydrogenases have been reported.The molecular mechanism of the reductive cyclization reaction catalyzed by OX4/CftD and the substrate selectivity mechanism among OX4 and CftD have also not been clarified.Based on the above research background,this thesis carried out the following three aspects of work:1.Crystal structure of alcohol dehydrogenase OX4 ternary complex and apo CftD.Through protein expression and purification,protein crystal screening and optimization,and crystal X-ray diffraction data collection and processing,we have got four OX4 ternary complexes,including OX4-NADP+-3-deOH ATC(2.3 ?),OX4-NADP+-3-deOH ATB(2.8 A),OX4-NADP+-3-deOH HSAF(2.6 A),OX4-NADP+-Y1(3.2 ?).We also have got one apo form crystal structure of CftD(2.0 A).Structural analysis revealed that OX4 and CftD belong to nonzinc-dependent alcohol dehydrogenase,each subunit contains a catalytic domain and a nucleotide binding domain,which is separated by a hydrophobic cleft.The substrate and NADPH in the OX4 ternary complex bind like a T-shape in the above cleft.2.Catalytic mechanism of alcohol dehydrogenase OX4.1)By identification residues that may locate NADPH in OX4 and evaluation of the function of NADPH-related OX4 mutants in vitro,we proposed that residues G169 and K195 may determine that OX4 prefers to use NADPH.2)By analyzing the crystal structure of OX4 ternary complex,we found that the hydride-transferring nicotinamide group of NADPH was close to C21 of the substrate,which provided structural support for the regioselectivity of OX4.3)By comparing the structure of the ternary complexes of OX4 and testing the function of the substrate-related OX4 mutants in vitro,we found that neither hydride transfer nor proton supply in OX4 reaction required amino acid residues to mediate,thus protons may come directly from the solvent water and amino acid residues(except W260)in the substrate binding pocket may affect enzyme function by affecting the binding of substrate to protein.4)Because both OX4w260F and CftDw261A can only catalyze the reduction of the double bond,along with the possible structure of the intermediate,the conserved tryptophan was proposed to be reapongsible for stabilizing the enol-like transition state by forming a hydrogen bond with the carbonyl group at C-1.5)Through comparison of the structure of the OX4-NADP+-3-deOH ATB and OX4-NADP+-3-deOH ATC ternary complexes,it was found that the different configuration of the substrates resulted in different protein-substrate binding modes,which may lead to the substrate stereoselectivity of OX4.3.Mechanism of the selectivity of OX4 and CftD for the polycyclic system of the substrates.Through comparison of the protein structure and sequence,three amino acid loop regions were identified that may affect the selectivity of OX4 and CftD for the polycyclic system.Then,loop exchanged OX4 and CftD mutants were constructed,and the function of them were evaluated in vitro.The results showed that the selectively of CftD mutant C-3 was transformed to be consistent with OX4,indicating that the amino acid loop291-300 in OX4(loop292-301 in CftD)are probably involved in the substrate selectivity of OX4 and CftD.In summary,based on the previous biosynthesis of PoTeMs,this thesis revealed the mechanism of the reductive cyclization reaction catalyzed by PoTeMs related alcohol dehydrogenase and their substrate selectivity based on the structural biology studies of OX4 ternary complex and apo CftD,and in vitro function evaluation of the related mutants.This study not only provides experience for the structure acquisition of similar proteins,broadens the understanding of the catalytic mechanism of alcohol dehydrogenase,but also provides structural and theoretical basis for engineering of the polycyclic system of PoTeMs. |
