| The rule of structural and dynamic change of industrial enzyme during catalytic reaction,which is hard to decipher,play a critical role in bio-catalysis study.Fungal lipases,for example,Rhizopus lipase,as one of the most important branch of lipase family has become widely applied industrial enzyme benifit from its structural complexity and functional diversity.However,the incomplete of catalytic mechanism like prosequence structure and lid dynamic usually results in low efficiency of lipase engineering.X-ray diffraction can be used to solve protein crystal structure,yet not capable of providing dynamic information.Meanwhile,nuclear magnetic resonance(NMR)technique is powerful in protein dynamic study in solution.However,low spectrum resolution of larger proteins(>20 kDa)creates unique challenge for isotope-labeling and resonance assignments,which is required for structural and dynamic studies.Crystal structure provides space information for resonance assignment of large proteins.Hence,rational design based on lipase structure and dynamic solved by the combination of X-ray diffraction and NMR technique is of great significance to expand applications of lipase.Here we solved the crystal structure of a 33 kDa protein-Rhizopus chinensis lipase(RCL)as well as solution conformation and dynamic using NMR technology profit from efficient expression of isotope-labeled RCL in Escherichia coli(E.coli)and sufficient assignments aided by X-ray crystal structure.Moreover,structural analysis and mutation validation helped reveal the role of N-terminal prosequence.The activation mechnism of was revealed from comparison between the 2D spectra with and without organic solvent.Furthermore,possible solutions were proposed to express ILV-labeled protein in P.pastoris based on flux ratio analysis of Pichia pastoris(P.4pastoris)with and without precursors.(1)To fill up the blank of Rhizopus lipase prosequence structure and provide space information for resonance assignment for NMR study,by optimization of crystalization,a 0.06×0.05×1.5 mm single crystal of RCL was obtained.X-ray diffraction data were collected to 2.0 A resolution and the crystal structure was solved by molecular-replacement using Rhizopus niveus lipase(PDB entry 1LGY)as a search model.Preliminary structural analysis showed that RCL is a monomer with typical ?/? hydrolase fold of lipase family members.The catalytic triad residues are S172,D231 and H284 and oxyanion hole is composed of T110 and L173.The "lid" region,residues G109 to T123,is a short a-helix linked to the "core" of the protein structure by polypeptide hinge residues.Three disulfide bolds are formed from 7 cysteines.The density map of residues T24-G31 was missing while residues D1-S23 present high B-factors.(2)Many key proteins needed for biomolecular NMR studies require eukaryotic expression hosts for protein sample production.Pichia pastoris is a widely applied system for eukaryotic protein production,however,incapable of expression 2H,13C,15N;1H-Ile ?1,Leu-?,Val-?-labeled(ILV-labeled)protein to improve spectrum quality for large proteins,which produces great limitation as an expression host for NMR study.To address the difficulties of using P.pastoris to express isotope-labeled eukaryotic protein and study the solution structure and dynamic of RCL by NMR,13C,15N-labeled RCL was expressed in P.pastoris with expression level of 26 ± 3 mg·L-1 and labeling rate 84 ± 3%.Whereafter,2H-labeled RCL was also expressed to a level of 4.5 mg·L-1 and labeling rate of 59%.Furthermore,flux ratio analysis of P.pastoris with and without precursors Val,a-KB and a-KIV elucidated two major bottlenecks for the preparation of ILV-samples for structural biology in P.pastoris:the lack of a-KIV transport into the cytoplasma and the low efficiency of a-KB transport into the mitochondria.However,Val was efficiently deamidated to a-KIV by cytosolic branched-chain-amino-acid aminotransferase(BCAT)to sythesize Leu.We proposed two suitable solution to express 2H,13C,15N;1H-Ile ?1,Leu-?,Val-?-labeled protein in P.pastoris:strain with newly engineered a-KIV/a-KB transporters or with threonine dehydratase gene knocked out.(3)Using E.coli to produce ILV-labeled eukaryotic protein remains of big challenge in NMR study.To build up an efficient protocol for using E.coli expression system to produce isotope-labeled protein especially proteins with multiple disulfide bonds,and assign chemical shifts of ILV-labeled RCL,we combined E.coli strain BL21 trxB(DE3)and maltose binding protein(MBP)-fusion tag to produce 13C,15N-labeled RCL with final expression level of 15 ±2.5 mg·L-1 and labeling rate of 99 ± 0.2%.It formed the same disulfide bonds as that expressed in P.pastoris.2H,13C,15N;1H-Ile ?1,Leu-?,Val-?-labeled RCL was epxressed at a level of 4.3 mg·L-1.Final number of assignments for backbone and ILV sidechain were 229 and 83.These resonance assignments have been validated and deposited in the BioMagResDB(BMRB ID 26923).(4)Structural change and dynamic is the key problem to be solved in catalytic process.RCL solution structure and dynamic were identified using 15N relaxation dispertion experiment,Nuclear Overhauser Effect(NOE)and Residual Dipolar Couplings(RDC).The missing peptide T24-G31 of RCL was characterized as random coli and T4-A21 residues showed secondary structure and interactions with core protein.NOEs between prosequence,lid and core indicated prosequence binds near the lid and active site and also amplied the closed form of the lid in solution.The hinge region and the residues pointing out side of the active site showed high flexibility which also suggested that the lid is closed in solution.The RDC results showed a two-state model of closed and open lid conformations,with conformational interconversion on the microsecond timescale.The prosequence didn't increase the surface hydrophobicity of RCL but formed salt bridge,hydrogen bond and hydrophobic interaction with the lid and active site,which may affect the geometry and microenvironment of the active site and stablize the lid,which may affect the catalytic properties consequently.Mutation of the key sites in the N-termianl prosequence confirmed the role of N-terminal poly peptide:first is affecting interfacial activation by interacting with lid residues,second is changing the active site geometry by interacting with binding pocket reidues.(5)The effect of organic solvent on lipase properties is still inconclusive.To address the mechanism of lipase activation by organic solvent,we measured the kinetic parameters of RCL with organic solvents.KM of RCL increased markedly when the concentration of the organic solvents is over 20%.The kcat values and the catalytic efficiency increased as organic solvent concentration increase.Comparing the 2D spectra of RCL with 20%organic solvent and in buffer indicated stable structure of RCL.The peak heights of most residues reduced with organic solvents especially with methanol,ethanol,isopropanol and DMSO compared with acetone,which was also consistent with the kinetic results.Residue V121 in the hinge region of the lid was the most affected residues among these organic solvents,indicating the high dynamic the lid region with organic solvent.Chemical shift perturbation was observed both on the surface and inside the protein.Chemical shifts of residues G6 and L12 in the prosequence,G109 and M120 in the hinge region of the lid and G174 in the active site were the most dramatic,which proved that the lid was more open when adding organic solvents.All these results clarified the activation of organic solvents to RCL.It not only increases lid dynamic but also drives the lid to a more open confirmation to motivate catalytic reaction.And when concentration reaches a certain level,organic solvent molecule may cause competition with substrate thereby decreases activity. |
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