The Study Of Function Expansion And Mechanism Analysis Of The Cytocherome P450 Enzyme PikC Mediated By Non-canonical Amino Acids

Macrolide antibiotics are a class of broad-spectrum antibiotics that act as effective protein synthesis inhibitors and have significant therapeutic applications in the treatment of infectious diseases,respiratory diseases,and gastrointestinal diseases.PikC is the only P450 enzyme existing in the biosynthetic pathway of pikromycin,which is the first naturally occurring ketolactone discovered.Through enzyme and substrate engineering,the substrate versatility and product diversity of PikC have been greatly expanded.To our knowledge,no PikC mutant has hitherto been reported that can directly oxidize the precursor compounds of YC-17 and narbonycin,namely 10-deoxymethonolide and narbonolide.In order to achieve the oxidative modification of these macrocyclic lactone frameworks.which lack any anchoring groups,and to develop a method for obtaining structurally diverse macrolide compounds,the following experiments were conducted:1.Construction and activity analysis of non-natural substrate active enzyme PikCH238pAcFFirst,we performed semi-rational engineering of PikC directed by combining the existing crystal structure information of PikC and the structural characteristics of non-natural substrate molecules,using the genetic code expansion technology based on non-canonical amino acids.The results showed that PikCH238pAcF is the only mutant that exhibited catalytic activity towards both non-natural substrates,with substrate conversion rates of 17.9%and 24.7%for 10-deoxymethonolide and narbonolide,respectively.Moreover,compared with the wild-type.this mutant showed 0.9-and 0.7-fold increase in substrate conversion rates for the natural substrates YC-1 7 and narbomycin,respectively,and also changed the regioselectivity of the hydroxylation reaction of YC-17.Importantly,the non-natural activity displayed by the H238pAcF mutation cannot be reproduced by any proteinogenic amino acid at the same site.This mutant not only achieved oxidative modification of the macrocyclic lactone skeleton without any anchoring groups but also enabled the activation of inert C-H bonds in substrates with more stringent structures.2.Crystallography and mechanism analysis of PikCH238pAcFTo gain a deeper understanding of the structural basis underlying the catalytic properties such as non-natural activity and regioselectivity of the mutant(PikCH238pAcF),we conducted analyses on four crystal structures of the enzyme in both substrate-free and substrate-bound states,as well as molecular docking experiments.The analysis results indicate that due to the introduction of pAcF,the crystal of PikCH238pAcF stabilizes the catalytic conformation of the substrate through pAcF-mediated hydrogen bonding and conserved hydrophobic interactions.In addition,the introduction of pAcF fine-tuned the catalytic conformation of the two natural substrates and changed the regioselectivity of YC-17 catalysis.In addition,the introduction of pAcF broke the balance between the two hydroxylation sites of YC-17 and the distance between the active center,thus changing the regioselectivity of YC-17 catalysis.3.Construction of specific glycoside hydroxylaseGuided by various crystal structures of PikC and the results of single-point mutagenesis,we obtained a specific hydroxylase(PikCH238pAcF/E85Q/E94Q)for 10-deoxymethynolide and narbonolide through multi-site combination mutations of PikC.It is worth noting that we unexpectedly obtained a mutant(PikCH238pAcF/E85Q)that showed increased hydroxylation activity for both 10-deoxymethynolide and narbonolide during this process.4.Construction of artificial enzyme cascade reaction of unnatural macrolidesAfter optimizing the P450 enzyme-catalyzed reaction conditions,the mutant PikCH238pAcF/E85Q showed substrate conversion rates of 75.0%(10-deoxymethynolide)and 78.0%(narbonolide)for the two non-natural substrates,respectively.Furthermore,by combining the mutant with UDP-dependent glycosyltransferase BSGT-1,we established an artificial enzyme cascade reaction for non-natural macrolide compounds in vitro and obtained glycosylated products that were not accessible through natural pathways.The establishment of the artificial enzyme cascade reaction for non-natural macrolide compounds provides more options for enriching the structural diversity of macrolide compounds and screening antibiotics with potential therapeutic activity.In summary,a semi-rational mutagenesis of a microbial natural product biosynthetic P450 enzyme via non-canonical amino acid incorporation has been achieved in this study.Based on the multiple structural information of PikC and the structural characteristics of two non-natural substrates,we achieved the oxidation catalysis of the macrolide skeleton without any anchoring group by constructing a non-canonical amino acid-mediated substrate binding strategy.Moreover,a series of non-natural macrolide compounds that were inaccessible through natural pathways were obtained by constructing an artificial enzyme cascade reaction for non-natural macrolide compounds in vitro using the active enzyme PikCH238pAcF towards non-natural substrates,and the glycosylation-oxidation order in the biosynthetic pathway of pikromycin was reversed.The P450 enzyme engineering strategy based on non-canonical amino acid site-specific mutagenesis and the mechanism insight obtained from detailed structural analysis of enzyme-substrate complexes can be applied to the construction of more glycosylation-oxidation coupled systems in the future.Furthermore,the modification of such biosynthetic pathways can be achieved in vivo through exogenous feeding or in situ biosynthesis of non-canonical amino acids.With the help of structurally diverse non-canonical amino acids,more enzyme reactions or cascade reactions are expected to be functionally extended to design and produce more natural product derivatives for drug development.