Study on Sulfide Monooxygenases:Discovery,Characterization and Structure-function Relationship


Supervisor:xu jian he yu hui lei


Degree Year:2018





Optically active sulfoxides are important chiral intermediates for asymmetric synthesis.They also constitute the structure of pharmaceuticals,such as proton pump inhibitors or prazoles,which are a group of drugs whose main action is a pronounced and long-lasting reduction of gastric acid production.Optically active sulfoxides are generally obtained by asymmetric monooxygenation of sulfide precursors.In addition to chemical oxidations,biooxidation approaches have been developed rapidly in recent years.Two kinds of monooxygenases,including Baeyer-Villiger monooxygenases(BVMOs)and cytochrome P450 monooxygenases(CYPs),can catalyze the oxidation of sulfides.This dissertation was focussed on the asymmetric monooxidation of sulfides catalyzed by BVMOs and CYPs,including three parts:1)genomic mining,purification and characterization of BVMOs with novel features;2)molecular engineering and structure-function relationship analysis of cyclohexanone monooxygenase from Acinetobacter calcoaceticus(AcCHMO);and 3)analysis of electron transfer mechanism in the catalytic process of sulfide monooxygenase P450smo.Part 1.BVMOs can catalyze the asymmetric oxidation of sulfides and have been studied extensively by a great number of scientists in recent years.Although BVMOs display broad substrate ranges,no native enzymes were reported of activity towards the asymmetric oxidation of bulky prazole-like thioether substrates.Thus,genomic mining using the sequences of EtaA(has abilities toward several bulky thioether substrates)and CHMONCIMB9871(has ability toward methyl phenyl sulfide(MPS))as templates was performed,then the catalytic abilities toward MPS and omeprazole sulfide(OPS)were tested.BoBVMO and AmBVMO with high activities toward OPS were discovered from Bradyrhizobium oligotrophicum and Aeromicrobium marinum,respectively.And AcCHMO from Acinetobacter calcoaceticus exhibited good activities toward MPS.Subsequently,the three enzymes(BoBVMO,AmBVMO and AcCHMO)were selected for purification and their biocatalytic properties were investigated in detail.Evolutionary relationship and sequence analysis revealed that the three enzymes belong to the family of typical Type IBVMO and they showed the highest activities(117,25 and 839 U/gprotein)toward MPS among the tested substrates.The activities of BoBVMO and AmBVMO toward OPS were 18 and 24 U/gprotein,respectively.However,the solubility and thermostability of them are poor.On the other hand,AcCHMO can not convert bulky sulfide substrates,but its solubility and thermostability are better than BoBVMO and AmBVMO.It has half-lives(t1/2n)of 20 h at 30℃,which is 4.5 and 3.7 fold to BoBVMO and AmBVMO,respectively.Part 2.AcCHMO was selected for further engineering of substrate specificity.A double mutation variant,AcCHMOM1(K326C/F432L),was identified,debuting a trace activity towards OPS in which the active sites adjacent to the substrate tunnel were modified by combinatorial active-site saturation testing(CASTing).Subsequently,using AcCHMOMI as parent,we performed iterative saturation mutagenesis in the core regions(substrate binding pocket,substrate tunnel,FAD and NADP+prosthetic groups)and error-prone PCR of full-length to improve its activity.An ingenious halo-based selection method were constructed which was based on the difference of solubility between the substrate(OPS)and product(esomeprazole).The finally variant AcCHMOM6 exhibited 30.3 U/gprotein activity toward OPS.The reaction catalyzed by purified AcCHMM6 for producing(S)-omeprazole achieved 96%conversion after 22 h,with 3 g L-1 substrate loading.Meanwhile,the change of activity toward four distinct substrates with varied size and structure,including cyclohexanone,MPS,5-methoxy-2-methylthio-1H-benzimidazole and OPS revealed an evolutionary route of AcCHMO.We also determined the crystal structure of AcCHMOM2,and shedding some light on elucidating the greatly altered substrate specificity,that is,the shape and size of substrate tunnel is the key factor to the change of substrate specificity.Part 3.In this lab,a sulfide monooxygenase(P450SMO)was previously cloned from Rhodococcus sp.ECU0066,with high activity and good stereoselectivity toward many sulfide substrates.P450SMO also showed very poor activity(<10-4 U/gprotein)on OPS.P450SMO belongs to Class IV CYPs which are known as "self-sufficient" P450s.However,the low electron transfer efficiency limit its application.Therefore,it is very important to reveal its electron transfer mechanism for further improving its catalytic performance.The crystal structure of P450SMO was solved successfully by our lab and Zhou lab of Shanghai Institute of Organic Chemistry.Based on the crystal structure of P450SMO,the structure-function relationship and dynamic conformational control of electron transfer in P450SMO were studied.At first,we measured the kinetic parameters of each step in electron transfer process depending on spectrophotometric method.The results shown that the electron transferred from FMN to heme through Fe2S2 domain was rate-limiting step.Combined with the crystal structure,we speculated that significant conformational changes of Fe2S2 domain maybe occurred to achieve the successful shift of electrons.Specifically,chemical cross-linking of proteins coupled with mass spectrometry analysis and single molecule fluorescence resonance energy transfer experiments verified our speculation,that is P450SMO showed an FMN-oriented Fe2S2 state("F-conformation")and an heme-oriented Fe2S2 state("H-conformation").The conformational dynamics of this protein are affected by the oxidation state of the different enzymatic cofactors(DTT,NADPH,or Na2S2O3).And a conformationally gated two-step electron transfer mechanism for P450smo is identified:The first step in the mechanism occurs in the F-conformation,which is mainly explored when the Fe2S2 cluster is in its oxidized form;upon reduction of the Fe2S2 cluster—the action that initiates the second step of our proposed gated electron transfer mechanism—P450smo switches to the H-conformation.