Catalytic Promiscuity Study On Thermophilic Acyl-peptide Releasing Enzyme From Sulfolobus Tokodaii(ST0779) And Its Mutants

With the development of green chemistry, enzymatic reaction attracts more attention for its ability of high efficiency, high specificity, mild reaction conditions and environmental friendly catalytic activity. As a sort of natural catalyst, enzyme has already been widely applied in industrial production. Recently, the enzyme promiscuity has become an evolutionary concept and showed a high prospective role in chiral drug synthesis. Nowadays, the enzyme promiscuity is novel and promising research field.So far, it’s been reported that several hydrolases belong to lipase family have catalytic promiscuity. ST0779 is a member of POP peptidase family. The POP family is newly studied and special subfamily of serine proteinase, which has a α/β catalytic domain similar to lipase. It’s been a long time for our group to study ST0779 from Sulfolobus tokodaii. ST0779 has a very good thermal stability. We reported ST0779 has the catalytic promiscuity, which means it is able to catalyze Aldol Reaction and Henry Reaction. In addition, it shows the best combination of yield and selectivity among the enzymes which catalyze this reaction.In this study, site-directed mutations on its catalytic triad and R525 residue were conducted to study the promiscuity mechanism. These mutants show different catalytic activity of Henry Reaction which would definitely help us to reveal the catalytic mechanism and the major reason of catalytic promiscuity of ST0779.The mutations were obtained from reverse PCR utilizing recombinant p ET-28a-ST0779 plasmid as the template. After Dpn I digestion, transfected E.coli DH5α with mutated plasmids. The DNA sequencing certified the successful mutations of S439 A, D523 A, H555 A, R525 V and R525 E. The expression E.coli BLP strains were then built. The optimal expression conditions of wild type and all the 5 mutations are as following: the expression temperature was 30 ℃, the IPTG concentration was 0.1m M and 8h expression. To purify the raw protein, 80℃ heating 30 min was sufficient to obtain protein production with purity larger than 90% and molecular weight is 63 k Da. Finally, freeze-drying was applied to gain protein powder. To measure catalytic activity, the catalytic system of Henry Reaction is 20 mg of enzyme, 4 ml of TBME(tertbutylmethyl ether) and 1 ml deionized water(20% v/v water content) containing 4-nitrobenzaldehyde(0.1 m M) and nitromethane(1.5 mmol). The products were analyzed with 1H-NMR spectra and quadrupole time-of-flight mass spectrometer. HPLC analyses were run on an Agilent 1100 series equipped with a UV detector and an Astec CHIROBIOTICR chiral column to determine enantiomeric excess or with a reversed-phase VP-ODS column to determine yield. The results showed the ST0779 mediated Henry reaction, the optimal reaction conditions were able to achieve 90–94% yield and 92–94% enantiomeric excess. The catalytic triad and R525 mutations had a dramatic impact on esterase and peptidase activity. The catalytic triad mutations didn’t impair the Henry reaction catalytic activity. With nitroethane or nitropropane as substrates, the R525 V and R525 E mutations reduced the yield by 15%. All the 5 mutations caused significant decreases of enantiomeric excess value with nitropropane as the substrate. To further investigate the mechanism of ST0779 promiscuity, the 3 dimensional structures of ST0779 and its mutations were constructed with software Modeler of Insight II by homolog modeling. The molecular docking was conducted with Auto Dock Vina software and the substrates were nitroethane, p-nitrobenzaldehyde and 2-Nitro-1-p-Nitrophenyl-1-propanol. The top 3 scored structure showed there are no catalytic triad residues involved in substrates binding.In summary, this study not only discovered the new function of ST0779 to catalyze one of the most important carbon-carbon bond reaction, Henry Reaction, but also help investigate the possible mechanism behind this interesting phenomenon. This study suggests there may exist a different catalytic pocket to catalyze Henry Reaction, will finally make contributes to discover and develop new enzyme promiscuity and the enzyme application on green chemistry.