Structural Insights Into The Substrate Recognition Mechanism Of Alcohol Dehydrogenase OX4 For HSAF Biosynthesis

HSAF(heat-stable antifungal factor)is a polycyclic tetramate macrolactam(PoTeM)isolated from the biocontrol agent Lysobacter enzymogenes C3,which has a distinct chemical structure and potent inhibitory activities against a wide range of fungal species.The previous study on the biosynthetic mechanism of HSAF showed that the alcohol dehydrogenase OX4 catalyzes the reductive cyclization of a 5-5 bicyclic substrate(3-deOH alteramide C)with a specific configuration through an extended Michael addition reaction to form 5-5-6 tricyclic product,but only catalyzes the reduction of the double bond of 5-5 bicyclic substrates with other configurations(such as 3-deOH alteramide B)without cyclization.Based on the biosynthesis of HSAF,this study carried out structural biology and biochemistry research on OX4 in order to clarify the recognition mechanism of alcohol dehydrogenase OX4 towards substrates with different configurations.Here we report the 2.3 A crystal structure of OX4 by X-ray crystallography,and the binary NADP(H)-bound(3.1 A),ternary[NADP(H)and 3-deOH alteramide C-bound](3.1 A)complex structures.We found that OX4 is a homodimer,and each monomer contains a nucleotide binding domain and a catalytic domain,NADP(H)and the substrate 3-deOH alteramide C are both located in the cleft between the two domains.Subsequently,we verified the binding sites through in vitro activity and binding affinity assays of the point mutants.The residues S168 and K195 play a key role in the binding of NADP(H),and residues Y50,D57,D67,T110,F111,M113,F115,W260,Q265,M280,F281,F290 and L292 define the substrate binding pocket.In addition,a new compound,3-deOH alteramide E,was identified as the reaction product of OX4w260f with 3-deOH alteramide C in vitro.Based on the crystal structures of the complexes and the enzyme activity of each mutants in vitro,we preliminarily speculated the molecular mechanism of OX4 catalyzed the formation of the 6-membered ring in HSAF.Furthermore,we obtained the full-length iPKS/NRPS protein with high purity and catalytic activity in vitro for the first time,and identified two new ortho-dialkyl-substituted aromatic acids from Verrucosispora sp.NS0172.In summary,we solved the apo structure and two complex structures of alcohol dehydrogenase OX4 and identified a number of amino acid residues that are important for the function of OX4.Our results lay the foundation for further elucidation of the substrate configuration recognition mechanism of the alcohol dehydrogenase OX4,and provides a theoretical basis for the engineering of other related enzymes.