Research And Application Of Aminoacyltransferase Engineering Strategies Based On Internal Cavity Geometric Characteristics

Internal cavities in proteins arise from unique atom packing defects driven by the hydrophobic interactions among side-chain groups.These cavities exhibit distinctive ubiquity and dynamic fluctuation characteristics.Currently,the engineering of internal cavities has diverse applications,including enzyme-substrate interactions,substrate selectivity and specificity modification,thermal stability enhancement,and the elucidation of folding and unfolding mechanisms.Therefore,it is important to explore the geometric properties,encompassing volume,shape,and spatial distribution,for protein modification.However,recent research has primarily focused on volume-related changes,such as creation from scratch and filling from large to small,neglecting shape and spatial distribution aspects.In this study,we developed an internal cavity engineering strategy based on high-pressure molecular dynamics simulation（HPMD）to comprehensively explore how internal cavity volume,shape,and spatial distribution impact the stability and catalytic activity of transglutaminase（MTG）andγ-glutamyl transpeptidase（GGT）,expanding its potential food industry applications.The main research contents and results of this paper are as follows:（1）The geometric properties and distribution trends of the internal cavities were analyzed based on the Protein Data Bank（PDB）.Initially,8000 sample sets were randomly selected from the PDB database based on the SCOP classification principle,including 2000αproteins,2000βproteins,2000α+βproteins,and 2000α/βproteins.The results of the histogram of the frequency distribution showed that most internal cavity volumes of the proteins were below 100（?）³,indicating a prevalent trend of high numbers and small volumes.The cavity shapes were categorized into three shapes based on inertia matrices,namely,cigar-like,planar,and spherical.The results showed the overall negative skew distribution of cavity shapes.Specifically,cigar-like cavities accounted for the lowest percentage at 66.80%,while planar cavities reached the highest percentage at 30.77%,with spherical cavities comprising a mere 4.66%.Subsequently,we employed the BRENDA database to create a validation set comprising 140 samples,including 66 samples representing the strong enzyme dataset（retaining high catalytic activity even after incubation at temperatures ranging from 30-90°C for 0.5 hours）and 74 samples representing the weak enzyme dataset（inactivated under the same conditions）.Statistical analysis showed that the cigar-shaped cavity content was 4.19%higher in the strong enzyme than in the weak enzyme,exactly approximating the low planar cavity content of 4.36%in the weak enzyme,suggesting that enzymes with superior properties generally have a higher percentage of cigar-shaped cavities and that a change in the shape of the internal cavity towards cigar shape may improve the enzymatic properties.The results of polar and nonpolar residue occupancy showed that the proportions of Gly,Ala,Val,Phe,Tyr,and Gln in cigar shape were 15.92%,11.69%,14.20%,8.89%,20.66%,and 9.76%,respectively,which were significantly higher than those in the planar cavity（P<0.05）.（2）Using MTG and GGT as experimental subjects,we developed HPMD-based internal cavity engineering strategies to explore the effect of internal cavity volume on the stability and activity of enzymes based on the natural local compression differences of proteins.The T_m values of Q39L,E292D,R57L,and Y198F in the MTG mutant were increased by 5.0,4.3,1.0,and 1.0°C,respectively.Among these,the key internal cavity volumes of Q39L,R57L,and Y198F decreased by 12.24,8.77,and 6.19（?）³,respectively.The T_m values of V555L,Y418F,and T150L in the GGT mutant were increased by 3.0,1.2,and 1.0°C,respectively.The key internal cavity volumes decreased by 14.46,6.36,and 5.76（?）³,respectively.Data analysis indicates Pearson’s correlation coefficients of 0.5517 for MTG and 0.5961 for GGT,establishing a significant correlation between the reduction in internal cavity volume and the enhancement of thermal stability.Furthermore,compared with WT,the specific activities of R57L,Y198F,and V31L in MTG at the optimal temperature were 50.58,28.89,and 27.01 U·mg^-1,which were increased by 107.94%,18.79%,and 11.06%,respectively.Similarly,the specific activities of M97F,Y418F,and V555L in GGT were18.34 16.72,and 8.49 U·mg^-1,which were increased by 149.68%,127.59%,and15.56%,respectively.（3）Based on the all-atom spatial coordinates of the cavity shells,Mathematica was used to construct an inertia matrix,introducing the parameter w to depict the geometric shape of the cavity.By combining the key residues that affect the shape of the cavity and the potential sites selected by cavity engineering,we engineered mutants to systematically explore the influence of cavity geometric shape on both enzyme stability and catalytic activity.The results showed that the specific activities at the optimal temperature of R57L and Y198F in MTG mutants were increased by 108.35%and18.78%,and the T_m values were increased by 2.0 and 1.0°C,respectively.Furthermore,the w values for Cav4 and Cav5 increased by 0.010 and 0.002,respectively.Similarly,in the GGT mutant,the specific activities of M97F,Y418F,M97Q,Y418Q,and Y418G were 18.34,16.72,13.86,12.47,and 9.57 U·mg^-1,respectively.These values represented increases of 149.5%,127.5%,88.6%,69.7%,and 30.2%compared to the WT,respectively.Correspondingly,the w values of each mutant cavity increased by0.007,0.010,0.017,0.013,and 0.005.The results indicated that the change of cavity shape to cigar shape could improve the catalytic activity of the enzyme.（4）Considering the spatial distribution of internal cavities and drawing insights from mutations that exert substantial influence on both cavity volume and geometric shape,we delved into the impact of multi-cavity geometric characteristics on enzyme properties.We designed 14 multi-point mutants for MTG and 19 for GGT.In the MTG mutants,the optimal mutant,F259W/R57L/Y198F,demonstrated a notable 3.3-fold,reaching 103.26 U·mg^-1,and the T_m value was increased by 3.3°C.The hardness of cross-linking gel protein was increased by 46.77%to 4.55 N.DSC results showed that the melting temperature T_m^H was increased by 3.9°C.SEM results showed that the structure of crosslinked gel protein was denser and mostly polygonal honeycomb structure.In the GGT mutants,the optimal mutant,Y418F/M97Q was increased by 4.8-fold,reaching 42.8 U·mg^-1,and the T_m value was unchanged.L-Theanine was synthesized by whole-cell synthesis in a 5 L bioreactor with a yield of 15.4 g·L^-1·h^-1.Mechanistic analysis unveiled that the combined effects of volume reduction and shape alteration in multi-cavity mutants induced spatial coordinate shifts,and broadened the catalytic activity centers,leading to improved substrate binding and release efficiency,ultimately resulting in catalytic efficiencies(k_cat/K_m)of 376.24 and 130.5 s^-1·m M^-1 for F259W/R57L/Y418F and Y418F/M97Q,respectively,which were increased by 3.5 and16.4-fold,respectively,compared with WT.In addition,computational analyses showed that the free energy of binding to the substrate was reduced by 55.70 and 30.00kcal·mol^-1,respectively,indicating improved enzyme-substrate affinity.