Molecular Evolution Of The Substrate Pocket Of Thermomicrobium Roseum N-demethylase

Demethylation is a key step in the transformation of many natural products into active forms.Due to the special properties of-C-N-bonds,N-demethylation has always been a challenge.At present,N-demethylation reaction usually requires extreme reaction conditions,and has some defects,such as long reaction time,cumbersome steps,toxic by-products,heavy metal contamination and so on.N-demethylase can remove the methyl group on primary ammonia or secondary ammonia,so as to realize the transformation of biological macromolecules or compounds.In this study,sarcosine oxidase(TrSOX)derived from Thermomicrobium roseum DSM 5159 with the potential for N-demethylation was used as the research model,and its crystal structure was solved.The natural evolution in the non-conserved residues of key microdomains—including the substrate pocket,and entrance site—were then identified using ancestral sequence reconstruction(ASR),and the substitutions that accrued during natural evolution were recreated by site-directed mutagenesis.On the basis of retaining the original stability,mutants improved the catalytic activity,expanded the range of substrate spectrum,and identified the enzymatic hydrolysis products.Major results are listed below:(1)TrSOX crystal structure analysis.Almost all the homologous enzymes sharing over 50%sequence identities with TrSOX are uncharacterized and it is necessary to obtain the TrSOX 3D structure.After analysis,it was found that TrSOX is a monomer,showing a typical PHBH structure,which is divided into a substrate-binding domain and a FAD-binding domain.The substrate-binding domain contains 4 α-helices and 9 β-sheets,and the FAD binding domain contains 12 β-sheets and 8 α-helixes connecting the sheets.(2)Study on evolution trend and activity modification of substrate pocket and substrate entrance.To track the evolutionary trends of key microdomains,we obtained TrSOX mutants through ancestral sequence reconstruction and site-directed mutagenesis to study their catalytic properties,substrate scope,and stability.Among them,E251V showed improvement of the catalytic activity of the original substrates and enlargement of the substrate scope.S320K showed the most effective promotion of the terminal N-demethylation of N-methyl-L-amino acids.The catalytic activity of the double mutant S320K/F243Y for N-methyl-amino acid substrates was further improved,and the catalytic activity of the triple mutants S320K/E251V/P61E and S320K/E251V/V174A for nitrogen-containing heterocyclic compounds was improved.It is worth mentioning that these mutants still retain the original enantioselectivity and stability.(3)Analysis and identification of catalytic reaction.There is no commercial standard for the catalytic reaction products of alkaloid substrates,so it is necessary to identify the reaction products.The reaction products were separated by TLC,and the structure of the reaction products was identified by LC-MS and NMR.The results showed that S320K/F243Y could completely convert most of the N-methyl-L-amino acids into demethylated products within 2 h without racemization.E251V can oxidize secondary ammonia in cyclic alkaloids to tertiary ammonia,but it is always inactive to its enantiomers,suggesting that this mutant is efficient in the enantiomeric separation of these racemic compounds.After reacting with E251V for 72 h,the conversion rates of N-methyl-1-naphthylmethanamine,1,2,3,4-tetrahydroisoquinoline and hexamethyleneimine all exceeded 90%,which was higher than that of other alkaloid substrates.