Alteration Of Substrate Specificity Of Thermophilic Esterase By Rational Design

Esterase can catalyze the transesterification of a large number of natural and unnatural ester substrates. These enzymes generally exhibit high enantioselectivity and widely applied in industry. Various esterases with excellent characteristics which are essential for application can satisfy the demand of production, especially those with high thermostability. However, esterases/lipases applied in industry usually lose their activity at harsh conditions, especially at high temperatures. Thermophilic acyl aminoacyl peptidases/esterases exhibit high stability and can catalyze hydrolysis of some esters. Therefore, they are perfect original enzymes for molecular engineering to obtain expected esterase/lipase with excellent characters.Protein rational design is a technique of protein engineering arisen decade years ago. Based on the understanding of protein structure, protein refining is realized by protein engineering techniques, and new function is obtained. Compared with directed evolution based on non-rational design, this method can obtain the expected protein more rapidly.The molecular mechanism of acyl aminoacyl peptidase/ esterase APE1547 from thermophilic archaeon Aeropyrum pernix K1 has been investigated in our previous research. Based on the analysis of sequence and structure, saturated mutagenesis has been performed on the conserved site R526, which is then considered as a significant evolutionary site for POP and HSL family. Therefore, it is considered that specificity of enzymes is relative. In the viewpoint of evolution, the specificity of enzymes may be evolved from the"enzyme non-specificity"or"enzyme promiscuity", while the enzymes with non-specificity should be the"primitive enzymes"that some enzymes evolve from. Obviously, acyl aminoacyl peptidase/esterase APE1547 can be considered as such"primitive enzymes"in evolution.In this paper, we used the idea of protein rational design to refine APE1547 by protein engineering method, and got a chimera which showed significant change in substrate specificity and affinity. First, AFEST from Archaeoglobus fulgidus and lip-1 from Candida rugosa were of difference in sequence and highly similarity in 3D structure with APE1547. The catalytic domains of the three enzymes were canonicalα/βfold, but the N-terminal showed great differences. The N-terminals of esterase and lipase were a small cap and a bigger lid, respectively, while that of APE1547 was an even biggerβ-propeller domain which got more than 300 amino acids. Many reports showed that the N-terminal domain played an important role in controlling the in-out catalytic center of substrates. For this reason, we swapped the propeller of APE1547 with cap and lid, respectively. Before the protein engineering, we did silico screen with the computer- assisted- design to get a reasonable site. After getting a series of proper chimera, we chose the best one in energy to design the experiments. The result of experiment showed that the substrate selectivity of AAM6 whose propeller was replaced by the cap changed from long chain to short chain. On the contrary, the Al with the propeller replaced by lid showed high affinity for long chain ester. In spite of great changes in structure, both the two enzymes had high thermostability as APE1547. Consequently we analysed the 3D-structure of lipase from different sources. It was revealed that lipases which could hydrolyze the hydrolysis of long chain substrate had an extra peptide near the catalytic center, though all the enzymes belonged toα/βfold superfamily. Further investigations showed that Asp/Glu of Ser-Asp/Glu-His catalytic triples in serine hydrolase family always located on this peptide. It was indicated that this extra peptide appeared not by chance, and interacted with a function conserved region. Interestingly, APE1547 lacked this peptide. Therefore, the peptide was inserted in the backbone of chimera AL. The newly constructed chimera"ALI"was soluble in the supernatant of cell lysate when expressed in E.coli, and showed the equal esterase activity with another chimera"AL". This data suggested that rational insert of a peptide would not influence the activity significantly.The significance of the work could be summaried as follows: peptides with different functions indeed can be used as"building blocks"to"transplant"between the proteins with low homology and similar structure. This may be one of the evolution mechanisms for protein molecular: inserting different peptides into the frame of primitive enzymes with promiscuity will introduce different functions, and then a protein superfamily with different function has formed initiatorily. By the following"subtle regulation"among the members of super family through mutagenesis in primary sequence, divergent evolution for function and sequence occurred among them. Second, enzymes with"promiscuity"can be used as the primitive enzymes for molecular evolution. With the primitive enzymes for protein engineering selected, guided by the rational design spirit, the combination of directed-evolution based on site mutagenesis and protein elements recombination technique will speed up the process of protein engineering, and then increase the resource of enzyme/protein with excellent characters significantly.