Engineering Substrate Specificity Of Acylaminoacyl Peptidase/Esterase From Archaeon Aeropyrum Pernix K1 By Semi-rational Design

Esterases and lipases catalyze a diverse array of esterolytic transformationson a large number of natural and unnatural ester substrates. These enzymesoften exhibit high enantio-and regioselectivities;however, there remains aneed for more diverse esterolytic and ester synthesizing enzymes, particularlythose with thermostability can work under special condition. Proteases(including amidases and peptidases) also catalyze ester hydrolysis (andsynthesis in nonaqueous media) and are well known to possess broad substratespecificities. In fact, proteases are more reactive on esters than on peptides. Soproteases, as a potential esterolytic enzymes, is important to deserve intensivestudies on this way.Directed evolution has rapidly emerged to be the method of choice for thedevelopment and selection of mutated enzymes with improved properties suchas improved thermostability, altered substrate specificity, increasedenantioselectivity, inverted enantioselectivity. Many research groupssuccessfully rely on whole-gene random mutagenesis and recombinationapproaches for the directed evolution of enzymes. Recent advances in enzymeengineering have used a combination of these random methods of directedevolution with elements of rational enzyme modification to successfullyby-pass certain limitations of both directed evolution and rational design.Semi-rational approaches that target multiple, specific residues to mutate onthe basis of prior structural or experimental knowledge create 'smart' librariesthat are more likely to yield positive results. Although protein engineeringhave accomplish such a impressive feats in optimizing the properties ofenzyme. But it have not provide comparatively help in improving ourunderstanding of origins of protein function and reaction mechanism. So anintensive mutagenesis study on hot-spot site in evolved mutants will shed newlight on structure-function relationship of protein.The putative APH gene (APE1547) from the thermophilic archaeonAeropyrum pernix K1 has been over-expressed in Escherichia coli, and therecombinant protein Acylaminoacyl peptidase/Esterase (APE1547) has beenpurified and characterized by us early. The enzyme shows an optimaltemperature at 90oC for enzyme activity and is extremely stable. Therecombinant protein showed hydrolytic activity for a wide range of substrates,including p-nitrophenyl alkanoate esters of varying alkyl chain lengths,pNA-labelled amino acids, and peptides. Of a series of Ac-amino acid-pNAstested, APE1547 shows the highest activity for Ac-Leu-p-nitroanilide(Ac-Leu-pNA). In an earlier study, the maximal esterase activity has beenobserved for the substrate p-nitrophenyl caprylate (pNPC8). Both optimumsubstrates have bulky hydrophobic side chains. Acylaminoacyl peptidase(APH) belongs to the prolyl oligopeptidase (POP, EC 3.4.21.26) family ofserine protease, which also includes dipeptidyl peptidase IV (DPP IV, EC3.4.14.5) and oligopeptidase B(OB, EC 3.4.21.83). The POP family is arelatively new group of serine peptidases and different from the classic serineproteases, trypsin and subtilisin, in several structural features and catalyticbehaviors. Compared with the classic serine protease, the members of the newfamily are more similar to lipase: (I) the members of POP family contain acanonical α/β hydrolase fold, and the catalytic triad is covered by an unusual7-bladed β-propeller;(II) the enzymes share the same sequence order ofcatalytic residues (Ser…Asp…His) with lipase, which is different from that ofthe well known trypsin (His…Asp…Ser) and subtilisin (Asp…His…Ser);(III)the POP family shares the same Gly-X-Ser-X-Gly motif with lipase. Polgár'sgroup has studied the structural and evolutionary relationship between thePOP family and the microbial lipases family by comparing the segment nearthe catalytic residues. There is no significant sequence homology betweenlipases and peptidases, except for a 10-residue segment near the catalytic Ser.However, these two families have similar catalytic triads and a partiallyopened active site. In fact, APH shows comparable peptidase and esteraseactivities;it can catalyze both the removal of an N-acylated amino acid fromblocked peptides and an acyl chain from esters. An understanding of thedecimations for the peptidase and esterase activities of APH is an interestingissue, which may provide us with more information about the molecularevolution of the POP family.In this study, firstly, thermophilic Acylaminoacyl peptidase/Esterase(APE1547) from Aeropyrum pernix K1 was subjected to directed molecularevolution to generate mutants with increased esterase activity. Based on itsextreme thermostability, a sensitive high throughput method was set up forscreening esterase activity of APE1547. Two successive rounds of randommutagenesis resulted in a variant that exhibits a 1.5-fold improvement ofspecific activity and a nearly 4-fold increase in expressed level, which resultedin 6-fold increase in total activity. Basing on the sequence and structuralanalysis of the evolved mutant, saturation mutagenesis on R526 site wasperformed on wild-type gene and further evolved mutants was obtained. Themutation library of R562X showed strikingly different effects on these twokinds of substrates. Nearly all mutants display a decreased peptidase activitywith Ac-Leu-pNA as substrate. More than 80% of the clones showed anactivity less than 20% of the wild-type enzyme. In contrast, they exhibitedhigher activity for pNPC8. More than 90% of the clones showed esteraseactivity higher than the wild-type enzymes. Meanwhile, about 12% of theclones have activity more than 5-fold of the wild-type enzymes. These resultssuggest that residue 526 plays an important role in determining the peptidaseand esterase activity of APE1547.Kinetic assays revealed all of the R526X mutants have reduced peptidaseactivity as measured by Ac-Leu-pNA hydrolysis. Generally speaking, thereduced activities are due to the significant increases in Km rather than kcat,although the kcat of R526E is dramatically decreased. The Km of R526A ishigher than the mutants with bulky hydrophobic side chains such as R526V,R526I, and R526L, suggesting larger bulky hydrophobic side chains play asignificant role in substrate binding. The charged mutants at this positionchanged the Km most significantly;R526K and R526E had 25-and 14-foldincreases in Km, respectively.All mutants showed an increased esterase activity with substrate pNPC8,except that the R526E mutant decreases the activity slightly. The increase inesterase activity of the mutants is attributed to the increase in kcat since the Kmis in the same range as the native enzyme. These results indicate that thesubstitutions at this position affect the catalysis but not the substrate binding.The large hydrophobic mutants (Ile, Leu, and Val) increased the catalyticefficiency by 3.4-to 5.7-fold. R526K and R526A showed a small increase of50-60%.The R526X mutants showed increased esterase activity and decreasedpeptidase activity, respectively. Therefore, their specificity toward two typesof substrates has dramatically changed. The ratio of the catalytic efficiency(kcat/Km) between esterase substrate and peptidase substrate for each mutantbecomes much larger than the wild type. In the case of R526V, the esteraseactivity becomes ~150 times higher than the peptidase activity. A moredramatic effect occurred to mutant R526E, which essentially completelyabolished the peptidase activity but decreased the esterase activity only by afactor of 2, leading to a 785-fold of difference in the two enzyme activities.The above results unambiguously confirmed the importance of position 526 insubstrate discrimination and illustrate that enzymes can be evolved todiscriminate their substrates by a single mutation.The individual kinetic constants and the corresponding activationenergies for Ac-Leu-pNA hydrolysis were measured. The parameters forR526E could not be obtained because of the extremely low activity. Comparedwith the wild-type APE1547, all of the mutants have an obvious lower k1 forAc-Leu-pNA hydrolysis. For example, the R526A mutant is low in k1 by8-fold. Since k-1 is 1.4 times higher than the wild-type enzyme, the guanidinegroup of Arg526 is not only involved in substrate binding but also stabilizesthe bound substrate. R526V shows a 3.4-fold decrease in k1, and its k-1approaches that of the wild-type APE1547, suggesting that the hydrophobicside chain is not as efficient as the side chain of Arg to help Ac-Leu-pNA todiffuse into the active site.The individual kinetic constants and the corresponding activation energiesfor pNPC8 hydrolysis revealed all mutants showed increased k2. Since k2 is therate-limiting step in the catalytic mechanism, revealed by burst experiments,its increase promotes the catalytic rate significantly. The R526V shows thehighest k2 (Table III), suggesting that the hydrophobic residue at this positionis more favorable for the formation of the acyl-enzyme complex. Thus, theincreased catalytic efficiency of the R526V for pNPC8 is the combined effectof a 2-fold increase in k1, a 5-fold decrease in k-1, and a 4.7-fold increase in k2.Together, they lead to a kcat/Km that is 5.7-fold higher than the wild-typeenzyme. The R526A also showed a 2-fold increase in k1, which is the same asthat of the R526V. However, its k2 and k-1 are similar to those of the wild type,suggesting that only large hydrophobic side chains are able to stabilize thebound substrate and accelerate the catalysis. This conclusion is consistent withthe fact that the HSL family shows a strong bias towards large hydrophobicside chains at this position.R526 is highly conserved among APHs. The crystal structure of APE1547shows that R526 is at the active center and forms an ion-pair network withGlu88 and Arg113. The molecular dynamics simulations showed that R526 isinvolved in peptide substrate binding by forming a hydrogen bond between theguanidine group and the main chain carbonyl group of the substrate. As aprimary part of the S2-P2 interactions, any substitution at position 526 resultsin reduced activity for Ac-Leu-pNA hydrolysis, as observed in the activityscreening of the mutant library. The kinetic analysis of selected R526 mutantsclearly showed a significant increase in Km. These results suggest that theconservation of R526 in this position might be required for binding thesubstrate and for the stabilization of substrate. Surprisingly, R526K exhibitedan unexpected larger increase in Km. The molecular dynamics simulationsshowed that Lys526 formed a salt bridge with Glu88, but lost the S2-P2hydrogen bond between the substrate and the enzyme. The unique bidentatecoordination of R526 cannot be replaced by any other residues since it isinvolved not only in binding to the substrate but also in stabilizing the enzymeand substrate complex by forming the salt bridgenetwork(substrate-R526-Glu88-Arg113). The screening results in ourexperiment show that the bulky hydrophobic side chains (Val, Ile and Leu) atposition 526 resulted in an increase of esterase activity. They have favorableeffects on each step of the substrate hydrolysis, as illustrated by the individualrate constants of R526V. Compared with the wild-type enzyme, R526Vshowed a faster diffusion rate of the substrate into the active site, a more stableenzyme-substrate complex, and an enhanced acylation rate. The increasedcatalytic efficiency may be caused by the more twisted ester substrate, assuggested by the molecular dynamics simulation of the R526V mutant.Although the possible relationship between the POP family and themicrobial lipase family was suggested by Polgár in 1992, the direct evidenceof their evolutionary relationship is still not known. Since three-dimensionalstructures are more conserved than sequences under evolutionary pressure, acomparison of protein structures is more powerful than a sequence comparison.A 3D structure alignment by the combinatorial extension (CE) method(available on the World Wide Web at http://cl.sdsc.edu/) reveals that thecatalytic domain of APE1547 is surprisingly similar to that of AFEST fromArcheoglobus fulgidus of the hormone-sensitive lipase (HSL) family. Thecatalytic domains of APE1547 and AFEST share only 18% sequence identity,but the overall backbone deviations are less than 2.3 ?. Moreover, both of thefamilies show high activities for the long acyl chain ester. The structural andfunctional similarity between the two families clearly indicates they areevolutionarily related and might diverge from a common ancestor.In conclusion, we found that a single mutation at position 526 of APE1547can have very different effects on the peptidase and esterase activities withAc-Leu-pNA and pNPC8 as substrates. The substrate discrimination betweenesterase and peptidase appears to be associated with the polarity, volume, andconformation of substitutes, an important phenomenon that has not beenrecognized for the extensively studied POP family. This study provides thefirst direct evidence that the POP and lipase families are evolutionarily related;and R526 may be an evolutionary marker.