Structures,Functions And Catalytic Mechanisms Of Different Oligomers Of(S)-Carbonyl Reductases From Candida Parapsilosis

(S)-carbonyl reductase isoenzymes(SCR,SCR1,SCR2 and SCR3)from Candida parapsilosis can reduce prochiral ketones to chiral alcohols with anti-prelog characteristics.These isoenzymes belong to short chain dehydrogenase/reductases superfamily(SDRs),which mostly exist as homotetramers or homo dimers in solution,only a few existing as monomers.The variation of oligomerization states of SDRs will induce the change of their catalytic behaviors,but the catalytic mechanisms underlying the oligomerization states of these enzymes remains unclear.Thus,it is of great significance to study the relationship between the oligomerization states and the functions of these enzymes and to unveil the molecular recognition mechanism.In this thesis,we focused on(S)-carbonyl reductase isoenzymes(SCR,SCR1,SCR2 and SCR3)from Candida parapsilosis,through enzyme kinetics assay,X-ray diffraction,Cryogenic electron microscropy(cryo EM)and bioinformatics,we discovered the significance of the conserved tetramer interface in maintaining the active site structure of SDRs,determined the relationship between their quaternary structures and functions,and revealed the catalytic mechanisms of the tetramer interface in regulating enzyme activities for(S)-carbonyl reductase isoenzymes.These results not only foundamentally showed the relationship between the tetramer interface and the enzyme activity in SDR superfamily,but also provide a thorough foundation for the preparation of an multienzyme biocatalyst.Then main results of this thesis:(1)Recombinant C.parapsilosis/p CP-his6-scr2 homologously expressed(S)-carbonyl reductase 2(SCR2)and efficiently reduced 2-hydroxyacetophenone to(S)-1-phenyl-1,2-ethanediol(PED).The heterologous gene of scr2 was homologously recombined into the genome of C.parapsilosis.Through optimization of fermentation time,at 36 h,an efficient biocatalyst was obtained.Through optimization of whole-cell biotransformation conditions(p H5.5 and 35 ?),recombinant C.parapsilosis/ p CP-his6-scr2 can efficiently biotransform 2-hydroxyacetophenone to(S)-PED with an optical purity of over 99% and a yield of over 99%in 1 ml scale.The optimized condition was then applied to a preparative scale(500 m L)reduction of 2-hydroxyacetophenone with an optical purity of over 99% along with an isolated yield of 70%.(2)Recombinant C.parapsilosis/p CP-sy-his-scr2 extracellularly expressed SCR2,protein-protein interactions between(S)-carbonyl reductases were discovered,and heterotetramer of SCR2-SCR was successfully prepared.Attachment of signal peptide and propeptide sy at the N-terminal of SCR2 enables the protein being secreted into the culture supernatant.The characteristic fragments of SCR2 and its related isoenzymes(SCR,SCR1 and SCR3)were detected in the purified supernatant from the fermentation supernatant of recombinant C.parapsilosis/p CP-sy-his-scr2 by MALDI-TOF-MS.Protein-protein interactions between SCR,SCR2 and SCR3 were demonstrated by pull down experiemnts.Heterotetramers of SCR2-SCR were prepared by heterologous co-expression of SCR2 and SCR in Escherichia coli/BL21.(3)The necessity of oligomeric interactions in maintaining SDRs active site structure is revealed.Crystal structures of apo SCR,binary complexes of SCR with NADPH,ternary complexes of SCR with NADP and ethyl 4-chloroacetoacetate,and binary complexes of SCR2-SCR with NADPH,which are all symmetric tetramers,were solved.SCR is shown as a symmetrical tetramer,whereas C-terminal his-tagged SCR(SCR-his6)was semi-dissociated and thus shown as an asymmetrical tetramer.The structural comparison between SCR and SCRhis6(PDB ID 3CTM)revealed the differences among the tetramer interface helices(T122-D144 and S172-L197).Combined with enzyme kinetics data,crystallization data and cryo EM data,the necessity of the symmetrical tetramer interface in maintaining the active site structure of SDRs was demonstrated as well as the prevalence of this conclusion to the other SDRs family members through data mining and structural analysis.(4)Point mutation of SCR-his6/A193 E was rationally designed to obtain dimeric enzyme,and the influence of SCR oligomerization states on the enzyme activities was revealed.The size exclusion and static light scattering(SEC-MALS)experiments proved that about 76% of SCRhis6 was dissociated into dimers and about 24% remained in the form of tetramers under the enzyme activity assay condition at p H 6.0.Based on crystal structure analysis,mutants of SCRhis6/A193 E,SCR2-his6/A193 E and SCR2/A193 E were rationally designed to obtain dimeric enzymes in solution.The enzyme activity assay showed that dimeric enzyme of SCRhis6/A193 E,SCR2-his6/A193 E and SCR2/A193 E were completely inactivate towards the substrates of 2-hydroxyacetophenone and ethyl 4-chloroacetoacetate which indicated that tetrameric enzymes dissociating to dimeric enzymes was the main reason for reduced enzyme activity of SCR-his6.(5)Exploration and deduction of the molecular mechanism underlying the different enzyme activities from symmetric tetramer and asymmetric tetramer.According to the data from steady-state intrinsic fluorescence spectroscopy,trptophan fluorescence anisotropy was calculated as well as the dissociation constants(KD)between SCR oligomers and NADP or substrate ethyl 4-chloroacetoacetate.The KD of SCR-his6/A193 E towards NADP is twice of that of SCR,while 8 times of that of SCR towards ethyl 4-chloroacetoacetate.Thus,SCRhis6/A193 E not only can bind to cofactor NADP and ethyl 4-chloroacetoacetate,but also had similar dissociation constants with SCR towards cofactor NADP and ethyl 4-chloroacetoacetate.Interestingly,trptophan fluorescence anisotropy data in Perrin plot showed no obvious change in buffer with different concentrations of glycerol for both SCR-his6/A193 E and SCR,which means that the apparent anisotropy values were from segmental motions.Together with structural information(T122-D144 and S172-L197)and Perrin plot data,it indicated that the apparent anisotropy differences should be mainly from two trptophans: W123 and W137,which partially belong to the tetramer interface helices.The different rotational diffusions of the residues on the tetramer interface(e.g.W137)will influence the rotational diffusions of the related residues(e.g.W123)close to the active site pocket,finally regulating the enzyme activities.