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The Nanobiotechnology Handbook

Yubing Xie

Artificial Enzymes

Publication details https://www.routledgehandbooks.com/doi/10.1201/b12935-5 James A. Stapleton, Rodriguez-Granillo Agustina, Vikas Nanda Published online on: 16 Nov 2012

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However, someproteins are stableandfunctionalinorganic environments (Klibanov2001), advantage of the range of hydrophobic/hydrophilic properties of the 20 natural amino acids. et al.1961).Water isthesolventfor alllife,andprotein folding andfunctiontakeextensive flexible three-dimensional structures determinedbytheiraminoacidsequence(Anfinsen polymers ofaminoacidsthat fold(inatypeofintramolecularselfassembly)into precise but ture andpressure. Proteins, thedominantclassofcatalyticbiological molecules,are linear nanoscale. These machines self-assembleandfunction in water atenvironmental tempera examples of “soft” nanotechnology from which wecanlearnmuchaboutdesignonthe tion tocatalysisperform structural, regulatory, andother centralroles inbiology, are natural logical macromolecules that catalyze chemical reactions. Biological machines, which in addi research thathasseensignificantprogress in recent years.Enzymesare thesubsetofbio molded ground-up, or but blocks injection the larger from from built atom by atom. People, do exactly this. constructed blue are nottems carved are whales, redwood and trees biological which sys from machines molecular The massive parallelization. through matter specificity, in huge efficiency, gains affordability, and of quantities enormous and to process pays that strategy placement attention to the synthesis to provide of potential atom each the has 1981). (Drexler top-down the bottom-uplevel the rather from systems than to build Amaterials matter the of manipulation at theatomic describes nanotechnology definition, its purest In 3.1 References 3.10 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 Introduction CONTENTS In thischapter, wewilldiscussartificialenzymes,afascinatingandpromising area of Introduction Conclusions Perspectives and Enzymes Nonprotein Artificial Other Ribozymes Beyond Proteins—Artificial without Cofactors Novo Metal De of Enzymes Design NovoDe of Metalloenzymes Design Antibodies Catalytic No Parent with Natural of Enzymes Selection Evolution Design Directed Library Rational with EvolutionDirected ...... 64 54 65 53 55 47 49 59 57 52 62 47 - - - - Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 interested in the opposite the problem: in adoptinterested we what can predict a given will sequences perhaps are we enzymes, more 2008). (Zhang reasonable accuracy artificial considering In foldsthe of of fewer most proteins than years, recent and in folding prediction in progress great to make allowed researchers biology. computer of structural power Increasing challenges have clever and algorithms grand theis one and of adoptedbe difficult by extremely therefore agiven is sequence will that structure three-dimensional the Predicting minuscule. folds be can competing two between govern forces that The difference subtle, energetic folding are the and 48 information encoded in mRNA and synthesizes polypeptides with specific amino acid amino specific with polypeptides synthesizes and mRNA encoded in information for the reads nanotechnology. inspiration ribosome The clear another acid is subunits, of the1997NobelPrizeinChemistry fordiscoveringthemechanismof ATP synthase. consuming ATP togenerateaproton gradient. PaulD.BoyerandJohnE.Walker shared half the “open”state.Undercertain conditions,theenzymecanalsorun inthe reverse direction, the ADP andphosphate combinetoform ATP. Thecyclecontinueswithatransition backto binds the ADP andphosphatemore tightly. A final turn inducesthe“tight”state,inwhich are bound. A 120° rotation of the stalk changes the conformation to the “loose” state, which tein. Inthe“open”state, ATP from theprevious cycleisreleased andnew ADP andphosphate beta subunits(red) that,alongwiththree The rotation oftheasymmetricstalk(green) causescyclicconformationalchangesinthethree channel intheF via amechanicalrotary mechanism(Boyer1997).Protons passingthrough atransmembrane gradients intohigh-energy chemicalbonds.Itharnessesthephysicalenergy ofthegradient F catalyze are limitedbytherateatwhichreactant moleculescanreach thembydiffusion. cycle. Some enzymes do their work with such remarkable efficiency that the reactions they desired reaction. Whenthereaction iscomplete,theyrelease theproducts, ready foranother geometry andchemicalenvironment thatminimizestheactivationenergy ofthespecific precise target moleculesoutofthecomplexcellularmilieuandplacetheminprecise usually impossibleinwaterandreserved fororganic solvents.Inshort,enzymespick amino acidswithinabindingcleftcanmodifythe local environment toenablechemistry potential reactants andcutunwantedsidereactions toinsignificantlevels.Hydrophobic confer incredible specificityonenzymes,whichcandistinguishbetweennearlyidentical binding cleftanditssubstrate,thegeometricalconstraintsplacedonabound their pK attack. “Secondshell”sidechainsmodifythe properties ofthese“firstshell”groups, tuning Side chainsare preorganized inidealpositionstoabstractprotons orperformnucleophilic a newbondtoformorbendmoleculestrainenoughencourageitbreak. to occur. Theymightbringtworeactants intojusttherightgeometricproximity toallow Enzymes grabspecificsubstratesandholdthemintheperfectorientationfora reaction often sodelicateastobenearlyimpossibleunderstand,muchlessreplicate bydesign. proceed onglacialtimescales.Themechanismsbywhichtheyachievethesespeedupsare 2003). al. et (Kuhlman nature found in not yet 2003) al. et (Dantas structures well as as nature from adoptthat structures protein well. as front New have sequences generated made been on this been has progress great problem, design protein the as and known is This structure? three-dimensional target o −F Enormous numbers of possible conformations are available are of possibleEnormous numbers to long conformations polypeptide chains. The ribosome (Figure 3.1b), (Figure ribosome The nucleic of composed and protein machine a molecular Nature provides uswithcountlessexamplesofbeautiful,complexnanomachines.The Enzymes increase bymanyorders ofmagnitudetheratesreactions thatwouldotherwise 1 ATP synthase(Figure 3.1a)transformstheenergy containedintransmembraneproton a s and enhancing the stability of the catalytically active state. The close fit between a s andenhancingthestabilityofcatalyticallyactivestate.Theclosefitbetween o region of ATP synthaseratchetthecentralstalkofprotein in120°steps. α subunits(blue),makeuptheF ∼ 100 amino acids can now be predicted with now with acids predicted be can 100 amino The Nanobiotechnology Handbook Nanobiotechnology The 1 region ofthepro - Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 Artificial Enzymes Artificial quite different than those of any natural environment. To modify natural enzymes to enzymes To environment. natural of any natural those modify than quite different applications or health often are industrial of human demands the host and cells, of their However, cheese. and havebeer evolved enzymes requirements specific natural the to meet have away with provided to produce enzymes humanity valuable their and products like For domestication centuries, of biological organisms nanomachines. of source catalytic only our been has scratch, nature from to createof enzymes designer absence amethod the In 3.2 more general approaches. require will conditions any under process reaction desired any perform to enzymes of active rational site of creation artificial the redesign, type this generated be can by properties remarkably with [2007]). al. et different enzymes While (reviewedby mutating intuition even acid active by by asingle Toscano site chemical amino were awarded both ribosome. of for the studies Chemistry 1974 in The ribosome. 2009the Nobel the Prize and Medicine in Nobel Prize and Gallant specificityand (about1982])efficiency error peracids 1 [Ellis amino 10,000 of short of far the fall methods peptide synthesis chemical synthetic Bulk functions. precise andperform fold polymers that specifically of creation information-rich the direct able is ribosome, heritable to to the information genetic Thanks polypeptide chain. added are blocks one-by-one acid end of building to agrowing the Amino sequences. PDBs 2WDK 2WDL.) and (orange). of RNA from site, consists (Produced catalytic the including ribosome, of the half shown). than More (not molecule mRNA an around together clamp that subunit (green) (blue) asmall of alarge and composed is It chains. of polypeptide synthesis the catalyzes and mRNA from information reads that a factory is ribosome for and ADP. ATP PDBfrom 1E79.) affinities (Produced (b) The distinct with states three among cycle which the in changes conformational causes 120° steps in (green) stalk central of the Rotation thase. figure.) color for CD companion (See FIGURE 3.1 Often, the activity or substrate preference of a natural enzyme can be modified drastically drastically modified be can or substrate enzyme preference activity of the anatural Often,

Directed Evolution Directed (a) Biological molecular machines. (a) F The machines. molecular Biological (b) 1 domain of the F of the domain β domains (red), (red), domains o −F 1 ATP syn ATP 49 - Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 50 Within the lagoon a population of the is phage diluted continuous by flow is Within the and that must before flowing period to division waste. their shorter for than aresidence time remain they bacteriophage. Fresh advantage mutation rapid of the high cycle and rate life of 3.2b). (Figure methods of traditional continuous evolution Phage-assisted (PACE) takes evolution (Esvelt of proteins 2011), al. et labor-intensive discrete, to the rounds opposed as (Stapleton activities 2010). Swartz and enzymatic of for multiple-turnover awide select it variety as can promising extremely is particular, Tawfikand 1998) Griffiths haveenzymes. been adapted of active in IVC, selection the for 2011), (IVC; 2007), compartmentalization display mRNA vitro Szostak (Seelig and in and for Recently, binding. to selections however, yeast display including (Chen methods al. et but have limitations, restricted Pluckthun address these been 1997) and (Hanes partially display ribosome as such techniques vitro throughput in high Extremely pressure. selection alternative, to find undesired of tendency byways host cells and the the of avoidingthe mutant DNAintotransforming of efficiency by the convenient but limited popular are and are screens and vivo In capable selections improvedor screen enzymes. of identifying 3.2a). found (Figure is asuitable enzyme repeated until cycle is replicated mutated,are and evolution. providing mutants for anew of round directed This final applicationthe underthe conditions of fittest candidates deemed the The function. generated, parent is protein desired for tested candidate the each is on anatural and diverse A of selective a collection “library”) mutants based pressures. specific (called application-with nonnatural, of a fitness protein the determine that pressures selective replace evolution natural the humans Directed which alaboratory-based is in method were created. enzymes evolutionary the these by process which have mimicked engineers substrates, protein or on nonnatural environments nonnatural within optimally function lagoon. out of the avoid to rapidly phage enough washing infectious within of itself copies produce must Agene infectious. not ovals). are pIII without Newly phage produced (green bacteria the in aplasmid from stars) (blue protein pIII of the expression induce activity desired the with proteins mutant (colored Only circles). protein mutant mutations), x’s producing red with indicating ovals (black genome phage the within contained gene target the express which bacteria, the infects phage The rectangles). (blue phage containing rectangle) (black-bordered (tan ovals) into“lagoon” a flow Bacteria intervention. human without proceed to of rounds hundreds or dozens (b) allowing begins. cycle PACE the process, another automates and amplified, and isolated is gene the mutants, the fittest identifies selection or (colored Ascreen circles). proteins mutant into genes the translate and transcribe (right), bacteria which into (red x’s). (top) mutations transformed are diverse a library genes with mutant The generates (left) gene of aparent Mutagenesis selection. and mutagenesis of sequential rounds of discrete consists figure.) color for CD companion (See FIGURE 3.2 Recently, developed been has allows that continuous directed technique a phage-based selection high-throughput evolution acustomized directed Each requires project (a) E. coli E. cells flowinto cells a well-mixed volume a “lagoon,” termed where Directed evolution and PACE schematics. (a) Traditional directed evolution PACE evolution and (a) directed Directed Traditional schematics. (b) The Nanobiotechnology Handbook Nanobiotechnology The Escherichia coli Escherichia -infecting Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 high natural error rate error of phage natural high replication generates of adiverse random set mutations wash out lagoon. of harmlessly the The and new host cells to unable infect are but these produce also new phage, activity desired the phage. lacking encode that proteins Genes to produce cells infectious encode replicate that active genes proteins within harboring production to the of pIII, for Therefore, infectivity. aphagelinked phage required protein is activity enzymatic desired host. of The abacterial upon infection expressed is which a copy lagoon.the phage to the contain be gene modified evolved,to The of are within time order on the cycle of is 10 replicate and bacteria fast enough to avoid fresh washed out.infect being phage The life Enzymes Artificial mutant could that support (PCR) reaction DNA and chain shuffling (Stemmer 1994),isolationthe a in of resulting evolution parent for the random as served directed scaffold by error-prone polymerase designed rationally residues. mutations catalytic introduced targeted that along with This of metallo alignment sequence loops cofactor-binding derived metal substrate- a and and from relieve steric constraints, hydrolase was removed (Park to 2006). parent al. of enzyme et the domain C-terminal The evolution to introduce catalyze new that reactions. extend it of creation enzymes to the substrates it exist, target and may impossible be to natural the between intermediaries structural clear when of activity type of a substrateparticular the specificity for altering substrate (Chen corticosterone 2005). Zhao and However, effective be can strategy this while receptor estrogen of human substrate natural the gap between progesteroneand to bridge were structural used the mutant. desired promoter the Similarly, generating succeeded in steroids the testosterone T7 promoter the with atsequence −11 base the T3 position followed full on the by selection propagation. on ahybrid T3 for of promoter the activity promoter Selection consisting T3 promoter, the with promoter on that for activity did not selection support and phage For example, PACE the in study, T7 RNA polymerase showed wild-type the no activity substrate. substrate adesired and awild-type gap between structural substrates the span one or evolutionary more which multistep bridging to in use paths been has strategy effective One parent enzyme. the not present in activities with enzymes generating in however, afew cases, In new foldsentirely evolution or functions. directed succeeded has like changes large it for is introducing than tweaks optimizing suited for small better mutations many too evolution by atsequence introducing once. Directed is, therefore, of space, sequence parental the vastness to stray from the far too it in unwise is islands are adapts of it its However,demands new to environment. the proteins functional because parent enzyme the built that watchmaker” “blind same The enzyme. of the mechanism of for enzymes. awide variety alinkage such to establish of clever ways to think continuous evolution motivate engineers protein imaginative will to pIII production, benefits the of linked be can evolution that activities of enzymatic PACE While set. mutations before converging optimal to the same upon the limited is T3 promoter the different accumulated lagoons two in experiment, runs: identical parallel were able initially researchers to followintervention. The in mutational the taken paths GTP. PACE enabled up of to 200 evolution rounds over to occur 8days no human with ATP with T3 promoter the initiate or that rather CTP and than recognize polymerase that coworkersand power demonstrated the of PACE by evolving new versions T7 RNA of the by amutagenesis plasmid. enhanced Esvelt optionally be can gene and target the within Another approach to generating new activity combined rational design and directed directed and approach design rational combined Another to new generating activity or aboutthe information structure evolution no benefitDirected the has requiring of β min, faster than that of the bacteria and shorter than the residence the shorter than and bacteria of that the faster than min, -lactamase activity into a glyoxalase into (GlyII; II activity -lactamase E. coli E. α ligand-binding domain, 17 domain, ligand-binding β -lactamase (MBL) enzymes were inserted into the scaffold scaffold the into (MBL) were inserted -lactamase enzymes growth in the presence of presence a1.0 the in growth β -estradiol, and the final target target final the -estradiol, and μ g/mL concentration of αβ / βα ) metallo ) 51 ­ Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 novel artificial enzymes is limited by the requirement for a homologoustherequirement enzyme. by limited natural is enzymes novel artificial GlyII a into examplean solution of fordesign a the GlyII as superfamily provided and by structural MBL, belongs same to which the inspired study this were in modifications scaffold designed The cefotaxime. antibiotic lactam the 52 activity for the prositagliptin ketone indicated positions in the binding pocket that could that pocket binding the in ketone positions indicated prositagliptin for the activity no with enzyme protein) to aknown of atransaminase similarity on sequence based ture (Savile 2010). al. tical et Ahomology 3D model (model struc unknown of with aprotein pharmaceu antidiabetic of an sitagliptin, hydrogenation synthesis the step in asymmetric to replace rhodium-catalyzed ahigh-pressure, enzyme at createdan Codexis scientists active mutants in information. relative no interposition with to a control enriched be library acid acomputationally identities active in redesigned site, amino neighboring was shown to (Lippow 2010), al. between library et such positions. One linkages the maintained which acid identities at multiple amino between correlations to the preserve as so constructed be bled also by PCR can to of yield adiverse mutant collection libraries genes. Designed degenerate synthetic atsity into built oligonucleotides, site each then is assem are which (Jäckel 2010). pools al. et synthetic competent from sequences selecting Compatible diver 2009) al. removed et (Halabi be can statistically sequences or avoidedof these by entirely evolutionary the Bias from space history sequence of the to searched. be size down the allowed acids are at position, each to provide about narrowing amino which information proteins (Oteyetal.2006)andfungalcellulases(Heinzelman2009). shuffling. These methods have been used to generate high-quality libraries of P450 heme folded proteins andhavebeenshowntooutperformlibrariesgeneratedbyrandomDNA is leastlikelytodisrupt thestructure. Theresulting designedlibrariesare enrichedin 2004) protocols analyzestructural contactstodeterminepositionsatwhich recombination (Voigt etal.2002)andRecombinationasaShortestPathProblem (RASPP;Endelmanetal. contacts, loweringthefractionoflibrarymembersthatfoldsuccessfully. TheSCHEMA function. However, randomrecombination canintroduce clashesanddisrupt important of sequencediversityalready vettedbynature forcompatibilitywithaparticularfoldand based librarygeneration.“Sexual”recombination ofhomologousgenestakesadvantage application ofstructural datatotheselectionofcrossover pointsduringrecombination- active mutants. in mutant libraries to enrich methods rational using by limitations are these developing of to strategies address a Researchers variety difficult. improved, be very is can protein new activity entirely generating low starting levels the in considerable haystack the requires luck. addition, present at in In activities needle while a of mutants, numbers large random finding and approaches to screen rely ability on the active the siteat from far could positions that not have rationally. predicted been However, beneficial finds mutations often relationships, random mutagenesis screening function and evolution. ofof ignorance structure/ protein directed our addition In to circumventing knowledge astrength is or functional for of structural absence any requirement The 3.3 In an example of how artificial enzymes could revolutionize the chemical industry, couldchemical revolutionize the enzymes example an of howIn artificial generation library in of homologousMultiple used be can alignments sequence proteins A simple but extremely successful rational library design method has beenthe Directed Evolution with Rational Library Design Evolution Library Directed Rational with β -lactamase is remarkable, is application approach the -lactamase generation to the of of this β -lactamase. While the successful conversion successful the of While -lactamase. The Nanobiotechnology Handbook Nanobiotechnology The - - - - Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 tein as a nanotechnological substrate is by no means limited to natural environments. to natural limited substrate by is no means ananotechnological as tein stable 24 for more remained than conditions, enzyme the harsh solution), substrates in the to keep (required 40°C, 250 and of abioreactor: environment solvents 50% organic very unnatural the in to function ity excess of >99.95%, enantiomeric by an orders four and of magnitude abil the along with improved activity an in resulted conditions evolution realistic directed industrially under mutations four with hadant low toward that substrate. the activity Additional of rounds for mutagenesis. targeted be identified a vari Site mutagenesis saturation screening and Enzymes Artificial generates libraries too large to exhaustively screen, active proteins can be isolated be when can active to exhaustively large too proteins generates screen, libraries approach this While structure. to compatible be likely three-dimensional adesired with acids at site of each possible set to the those amino up merely to chance, restricting nism much approach as diversity possible. as leaves mecha This catalytic preserving the while to fold likely to sequences stable into library structures the to protein restrict over entire the or rational computational uses design new approach construction to library interesting An 3.4 design. of library rounds further inform and for function folding sites and critical tify sequence/activity adetailed form method that iden this landscape from result that fitness thatsequence.The copiousof fitness of the data a measure as pool was taken selected the in of sequence each abundance The selection. of deemed active amutant by afunctional library 2011). al. et (Hietpas members of those sequences the to identify was used sequencing Deep 2010), Ferre-D’Amare (Pitt and ribozyme RNA ligase an 2010), Hsp90 chaperone the and WW-domain (Fowler the protein to generateof landscapes al. technologies fitness et ing 100 of 2008). al. et fewer mutants than (Ehren screening and synthesis the requiring conditions, again gastric under enzyme of the stability the increased that endopeptidase approach 2007). (Liao al. et identified five A similar thermostability in tomutations prolyl 20-fold with improvements Kvariants providedtion proteinase enough data to construct mutants over construc 100 of of library rounds fewer two a total custom-synthesized than of number mutants. of of Testing mutation asmall each contributions performance to the the of homologousapart teasing then and alignments sequences of analysis via identified mutations of promising combinations of mutants containing collections small synthesizing cholesterol Lipitor drug, involves strategy (Fox DNA 2007). al. et synthesis-based Asimilar for the material production for process the starting of the industrial of an demands the consideration. ProSARto efficiently was used adapt to halohydrinmutants dehalogenase and are beneficial pool deleteriousmutations as identified removed are from mutations Newmutation regression. mutations added least-squares resolved are is to bythe partial 2003). of individual each effect the sequenced, and and ProSAR, In screened mutants are (Fox al. et (ProSARs)relationships developed engineering been applied has and to enzyme development, drug in sequence–activity on protein popular based method algorithm an relationship improve. quantitative to the synthesis structure–activity and Bying analogy of economics more DNAavailable, the is attractive as becoming sequenc screen are and high-throughput no when valuable particularly be can approaches Statistical mutations. of combinations optimal predict and function and of mutations individual on stability Statistical methods and machine learning are increasingly applied to isolate the effects applied to effects isolate increasingly the are learning machine and methods Statistical A particularly exciting new extension of these ideas is the use of next-generation use ideas the is new of extension these exciting sequenc A particularly Selection of Enzymes with No Natural Parent No Natural with Selection of Enzymes mM substrate.mM Even these under h, demonstrating that pro that h, demonstrating 53 ------Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 acter ateachaminoacidpositioncontainedproteins ofdiverseprimarysequence,most ably withwater. A librarydesignedtodictateonlythehydrophobic orhydrophilic char the solvent.Incontrast,surfaceaminoacidstendtobepolarorcharged andinteractfavor proteins typicallycontain hydrophobic aminoacidsthatpackwithinthecore tohidefrom hydrophilic interactions are dominant drivers of protein folding: the interiors of folded lections of protein sequences that adopt a defined three-dimensional fold.Hydrophobic/ applied. be can selection high-throughput percentage of ahigh folded in or extremely when results design an proteins library the 54 factor in enzymatic catalysis is the lowering of the activation barrier by stabilization of the of the lowering by the activation of catalysis stabilization is the barrier enzymatic factor in computer and dominant state suggest the theory that simulations Modern transition 3.5 accelerate that by much 2 reaction as as the enzymes was followedzinc-dependent yielding library by mutagenesis finally recombination, and random this from acids. Selection loops of 12 9amino randomized two and with scaffold of azinc-finger consisted protein 2007). study, latter Szostak the library In starting the and, more recently, capable RNA two (Seelig and molecules of enzymes ligating to select 2001) acids Szostak (Keefe and ATP-binding random of amino from 80 sequences proteins of 10libraries from proteins capable desired are methods vitro of selecting In method. high-throughput of enzymeswithsubstratebindingcleftssimilartothoseobservedinnaturalenzymes. ecules (Dasetal.2011). Scaffolds withdesignedcavities ofthistypemayallowtheselection cavities intoaselectedbundleresulted inapocketcapableofbindingsmallaromatic mol als orothercofactorsuponwhichtheyrely forfunction.Targeted mutagenesis to introduce Given thetendenciesoffour-helix bundles,itislikelythattheselectedenzymesbindmet especially remarkable consideringthecomplexityofenzymestheyreplace inthisstudy. but directly selectedfrom adesigned library, possessminimalenzymaticactivity. Thisis these simple,102-residue helixbundleproteins, whichwere neitherdesignednorevolved the cellprevents ruling outallalternativeexplanations,itseemslikelythatatleastsomeof formation withplasmidsencodingfourselectedsyntheticproteins. Whilethecomplexityof growth. Theauthorsthenshowedthataquadruple knockoutcouldberescued bycotrans demonstrating thatmutationofkeyresidues inthesyntheticproteins abolishedrescue of assays. Theauthorsgotoadmirablelengthsrule outalternateexplanations,including given the slow-growing phenotype would likely be below the detection limit of such lysates orpurifiedsamplesoftheseproteins, buttheverylowlevelsofactivityexpected of thestrainsonminimalmedia(Fisheretal.2011). Activity couldnotbemeasured incell 1.5 ×10 of activity (DasandHecht2007;Pateletal.2009). monoxide (Moffet etal.2001)andproteins withperoxidase, esterase,andlipaseenzymatic patterned librariesidentifiedheme-bindingproteins (Rojasetal.1997)thatboundcarbon these proteins hadordered, native-likecores (Wei etal.2003a,b). Screening ofthese binary- which foldedintothedesired four-helix bundlestructure (Kamtekaretal.1993).Manyof Binary patterningisasimplebutsurprisinglyeffective methodforgeneratingdiversecol An alternative strategy is to select from a less-restricted library with an extremely extremely an with library aless-restricted from to alternative select is strategy An The samegroup transformed27single-knockoutauxotrophic

Catalytic Antibodies Catalytic 6 patternedhelicalbundlesandisolatedtransformantsthatrescued growth offour 12 or more mutants. of display, mRNA these, One to select used been has × 10 6 The Nanobiotechnology Handbook Nanobiotechnology The -fold. E. coli strains with a library strainswithalibrary ------Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 reaction. Catalytic antibody generation antibody aknowledge-driven (Golynskiy Catalytic reaction. is method and any chemical for antibody virtually a catalytic should2005). contain libraries varied These 10 approximately Between reaction. state desired of the transition capable the of stabilizing antibodies raises of animals immunizations in antigen an molecule as this using then and astable TSA reaction desired of synthesizing the and Designing for antibodies: catalytic basis the forms strategy This for reaction. that enzymes candidates to good act as are reaction (TSA)analog statetransition a andof a desired stabilize bind specifically that molecules 2003; Garcia-Viloca 2004). al. et Hammes-Schiffer state and (Benkovic Therefore,transition Enzymes Artificial is the major component that defines the catalytic activity and specificity, in enzymatic and specificity,enzymatic major in componentthecatalytic the activity is defines that ligands) shell chelating (the coordinating first the which catalysts in metal traditional to opposed As function. desired the to obtain proper the environment within and geometry stable must correct scaffold, design protein cofactor place the the metal in the 2006). (Ragsdale chemistry transfer, radical and electron breaking, and bond forming as such reactions facilitating by functions awider to range of perform biochemical metalloenzymes so-called these cofactorsmetal 2006). elements (Ragsdale allows diversity inorganic of the chemical The thought to are incorporate enzymes 2007). al. et natural much 30% As as of all (Bertini life to sustain acids, would they required amino not reactions able be the to catalyze all by groups offered the chemical the only to active protein use If sites were constrained 3.6 2010).Seelig thebody in and (Golynskiy response immune an to elicit enzymes artificial other than have advantage, an however, vivo likely less in therapeutic are as agents, antibodies since 2009) al. et (Belogurov plasticity solvent-exposed and active sites (Xu 2004). al. et Abzymes fold 2010), (Golynskiy Seelig and immunoglobulin have therefore and and low flexibility single to the to accelerate restricted use are Abzymes reactions. enzymes natural strategies 2010). (Golynskiyrelease Seelig and one only of many is state stabilization Also, transition state, catalysis or product transition to blocking tightly the too bind that enzymes in result the bind to to efficiency.designed TSA, catalytic which could specifically are Abzymes 2010).(Golynskiy Seelig and of 2.3 rate enhancements achieve maximum Abzymes evolved highly enzymes. outperform natural cannot abzymes most tailored al. et 2002).(Nevinsky exists However,enzyme or artificial no natural theevenwhich decarboxylation, esters, for peroxidation,and cyclization, lactonization, reactions and hydrolysis including catalyze of transformations, aplethorathat amides of chemical 1986; al. et Tramontanohave 1986). al. et antibodies generatedbeen artificial then, Since rate the reactions of the to enhance ability the with antibodies clonal hydrolysisthe to carbonates. TSAs produce haptens of and as esters were mono used mechanism. reaction 2010),Seelig of a“good” of the construction since understanding adetailed TSA requires The design of artificial metalloenzymes is challenging. In addition to a providing challenging. is metalloenzymes of design artificial The lower their could that explain of design abzymes several the drawbacks are in There antibodies, or catalytic first The “abzymes,” were generated 25 agoyears catalyzeto 8 and 10 and De Novo of Metalloenzymes Design 11 different specificities are present in a human antibody repertoire (Hanson et al. et (Hanson antibodyrepertoire in a human are present specificities different × 10 8 s −1 versus 7 ×10 versus 19 s −1 for natural enzymes enzymes natural for ∼ 10 3 -fold (Pollack (Pollack -fold 55 - Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 sisted ofsisted amphiphatic four (Lusuccess al. et 2009).significant fieldlately has biologyseen this computational advances recent the structural in and with because metalloenzymes de novo on the focus scaffolds, of design we artificial will choices of greater more of approach protein latter the widely because been the has used scaffold. protein Although existing an active sites within novo functional or by creating be de designed can 2010). Roelfes (Rosati and interactions shell metalloenzymes Artificial first- as important as be can catalysis, even interactions and second-shell more distant 56 from each monomer (shown as sticks) serve as the ligands. the as serve sticks) (shown as monomer each from residue His one twoand Glu and five-coordinated, 1EC5); is of Zn Fe (PDB entry each instead of Zn presence the in solved was structure high-resolution The cluster. adiiron binds that dimer of ahelix-loop-helix consists figure.) color for CD companion (See FIGURE 3.3 3.3). 2000; al. et Figure active the site proteins, of one of these By modifying rationally (Lombardi DF1 of was protein termed helix-loop-helix two motifs consisted and original cluster; iron the adinuclear bind that 2002). al. et bundles four-helix are proteins These 2001; al. et 2000; al. et 2003; Maglio al. et DeGrado Lombardi and Marsh 2002; Summa the called de into 2007). al. novo et (Monien bundles four-helix engineered designed was also Hecht 2007; 1993; al. et Kamtekar 1997; Rojas al. et Wei 2003a,b). al. et Heme oxygenase activity and (Das catalyze heme chemistry peroxidase bind and that bundles ofnumber four-helix hydrophobic ofity and to polar de novo heliochrome of to that the a design residues similar aperiodic with sequences of coworkers binary-patterned and libraries combinatorial used togroups bound on heme based are have that synthesized been most de ofthen, novo the metalloenzymes Since heme proteins. of to that natural hydroxylase similar activity had aniline “heliochrome” The first artificial metalloenzyme was designed without wasthe aiddesigned of computers, and con metalloenzyme artificial first The Computational were de applied methods novo the of in proteins di-iron aseries design due ferri due α -helical bundles (Lu et al. 2009). As discussed earlier in Section 3.4, Section (Lu in earlier 2009). bundles al. Hecht et -helical discussed As (DF) proteins, inspired by natural dimetal proteins (Di Costanzo Costanzo (Di proteins dimetal by natural (DF) proteins, inspired α -helices bound to a heme group (Sasaki and Kaiser 1989). Kaiser and to aheme bound -helices group (Sasaki This Structure of a de novo metalloenzyme. The DeGrado group’s DeGrado The DF1 design of ade novo metalloenzyme. Structure The Nanobiotechnology Handbook Nanobiotechnology The - - Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 of 4-aminophenol and 3,5-ditert-butyl-catechol. The new design also exhibited improved exhibited 3,5-ditert-butyl-catechol. and new also design of The 4-aminophenol activemutations the site DF3 in was pocket, termed was able and oxidation to catalyze the Glythe mutations incorporated 2009). Gly al. (Faiella et also new design, which This introduced by destabilization the the of conformational goal with overcoming modified the protein. Morerecently, of DF1 turn destabilized interhelical was thesignificantly mutation to Glyincluded two a Leu the and residues Ala four chains, of two of the an in 10 a of presence atmospheric the oxygen oxidation in the with catalyzing of 4-aminophenol DFtet, to accommodate substrate, the capable were they avariant able of to engineer Enzymes Artificial been generated, the RosettaMatch program attempts to graft each of these constellations constellations of each these attempts to graft program generated,been RosettaMatch the has of candidate collection theozymes alarge Once designs. most promising the identify to used then are calculations state Quantum-mechanical reaction. of the transition the or “theozymes,” enzymes, to stabilize designed are theoretical abzymes, these Like active sites reaction. target idealized for computationally the and model disembodied Baker thefirst protocol stepthe of first, ( chosen is scaffold the In contrast to in approacheswhich a novelenzymes. protocol of creation for artificial the guanine. with enzyme wild-type was seven orders of ammelide lower that the of with magnitude than enzyme designed residue for asparagine activity. designed was important the However, of the activity the deviation (Csquare root alpha-carbon mean theto a matchedthedesign loop designed of configuration the revealed that enzyme designed of the structure molecule. Acrystal ammelide docked loop, residueoriginal position placed asparagine to hydrogen form in an a bonds with 2009). new loop, The the was residues at two which residues four length shorter than in ×10 2.5 deaminase guanine PNPA of early to those abzymes. similar hydrolysis kinetics with was able mutations to catalyze relative type wild three to only the containing enzyme pathway. reaction along the intermediate ahigh-energy to stabilize designed Adesigned generation, antibody active catalytic underlying to that the site was similar Using astrategy hydrolyzed that create anew p-nitrophenyl enzyme acetate (PNPA; Bolon Mayo and 2001). Overscaffolds. adecade ago, active to scaffold site an athioredoxin into was designed protein new active sites for on designing have existing incorporation into studies focused far, Thus design. complexity computational the of enzyme for taming method promising nanoscale. the at to control physics of ability our and chemistry and mechanism, enzymatic folding and of protein of test provides understanding goal our Holy ultimate the the Grail. This remains predicted as fold function and of will that problems, asequence to design ability classes the pragmatic limited evolution-based solving in haveWhile very successful methods been 3.7 2009). al. least (Faiella 50 et cycles active for at remained and to previous variants respect with stability thermodynamic In a series of recent breakthrough reports, David Baker and his colleagues have described have colleagues David reports, his described and Baker of breakthrough recent aseries In of human specificity the loopComputational changed of asubstrate-contacting redesign computer in speed, computation emerged increases a continued as to the has Thanks 3 De Novo Design of Enzymes without Metal Cofactors without Metal De Novo of Enzymes Design -fold (Kaplan DeGrado and rate 2004). enhancement However, which redesign, this α -RMSD) of 1 6 -fold favor in substrate, of a target (Murphy ammelide al. et Å. Point mutants confirmed that correct Å.that Pointplacement correct of mutants confirmed Figure 3.4 Figure ) is to design to design ) is 57 Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 58 kindly provided byBaker. David provided kindly were models enzyme the and theozymes the for (6).coordinates The experimentally tested and (5) identified are models promising (4). scaffolds resulting The protein complementary to matched are sites active these and rotamers, side chain the (3) by varying sites of active created is ensemble Next, an theozymes. possible different state, generating transition the around groups functional and side chains of different positioning the optimize and guide to used (2) are calculations QM intuition. chemical by identified are state transition the stabilize might that groups pathway. reaction state(s) of the Possible functional intermediate(s) key and transition the and identify catalyze, will enzyme new the that reaction a step (1)choose first to is The enzymes. Rosetta figure.) color for CD companion (See FIGURE 3.4 1 Ly TIM barrel O s OH Hi s Overview of de novo computational enzyme design protocol for the the for protocol design enzyme of de novo computational Overview As 4 3 O p Ex e pe enz D ar te ? e novo ensemble and rimen tificia oz sting Ac 5 ymes ensemble 2 matching ymes Sca tive sit l ta 6 ffold l Ty e r O Ly The Nanobiotechnology Handbook Nanobiotechnology The s + Se H O r 2 O Jelly roll O Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 elimination, a reaction for which no natural enzyme is known (Rothlisberger et al. 2008). al. et (Rothlisberger known is enzyme no for natural areaction which elimination, Kemp the performs that 2008) al. et enzyme (Jiang an a substrateand nature not found in for tested Finally, activity. and 100 around synthesized candidates are computational filtering. intuitive and further down narrowed through is matches scaffold 2007). al. et Rosetta@home (Das of theozyme/ set computers called The aproject through made network possible by of adistributed volunteers who provide personal to access their computationally extremely step is intensive is and matching This Data Bank. Protein the from taken acids onto structures protein ofscaffold of each afew aset hundred of amino Enzymes Artificial Unlike proteins, nucleic acid-based enzymes can be directly amplified andsequenced, amplified directly be can proteins, nucleicUnlike acid-based enzymes evolution experiment. artificial the selection round during of first for the used be will that RNArandom pool to initial producethe transcription amplificationvitro in by andPCR, Gold 1990) of aDNA random and of regions, synthesis constant consists molecule with and Szostak 1990; (Ellington ribozymes Tuerkand general methodology to create artificial DNA cleavedstranded that (as RNA ribozyme substrates). parental to the opposed The cleave RNA of molecule evolved to an specifically consisted vitro single- in selected and novel with properties. catalytic ribozymes 1989. in to create artificial efforts have chemistry there increasing then, in Since been Prize 1983; Nobel the with 1982). al. et Kruger was recognized discovery breakthrough This (Guerrier-Takada properties had catalytic but also information genetic carried only al. et ribozymes. of composed RNA are orwhether they DNA. on artificial only Here, focus we will on depending or deoxyribozymes, ribozymes on nucleic termed based are acids. These However, but not instead is protein-based is of biological class enzymes important another composed of those protein. far,So enzymes: we have of artificial reviewed major class the 3.8 enzymes. by artificial for mimicry target promising is and enzymes, by natural used strategies catalytic one most of important is reaction, the desired for the theand substrate key active site cost ofresidues fixing the optimal in orientations ances bal scaffold protein of the folding energy the which in group preorganization, Functional oriented were forpeptides entropic ligation, perfectly the reaction. cost of reducing the substrate of the activated catalyst, face. termini to the bound chemically When the binding 1996; 2001). al. et Saghatelian a peptide to was fold designed presenting The ahelix into 16-residuetwo al. et halves (Lee to C-terminal its N- own and corresponding peptides The 32-residueenzyme. peptide catalyzed replication ligation artificial theits own by of for scaffold adesigned an as early served ment. an example, In helix abinary-patterned application the and environ active for the site chemistry to optimal be designed scaffolds available any laboratory is and 2011). al. in et for (Richter use detail in described been has oneinto product, followed shortly (Siegel 2010). al. et procedure design enzyme Rosetta The substrate two combines molecules Diels–Alder which reaction, the catalyzes that enzyme one bonds, only substrate bind break therefore molecule. and An reactions of these Both The first enzymes designed using this method were retro-aldolases that break a bond in thatbond a break retro-aldolases method were this using designed enzymes first The The first artificial ribozyme was published in andwas published ribozyme 1990 Joyce(Robertson artificial 1990) first and The early independently the 1980s, RNA discovered notIn that molecules Altman and Cech activeinto sites Eventually,de novo incorporate artificial will design it hoped enzyme is Beyond Proteins—Artificial Ribozymes Beyond Proteins—Artificial 59 - - Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 dependent RNA-polymerases RNA of helix able an a complete are that to polymerize turn Pokrovskaya 1999; 1999; Ellington and Rogers Joyce Robertson and 1999) RNA- true and 1993; Szostak and 1995; al. et Ekland Ikawa 2004; al. et Jaeger 1999; al. et Landweber and are ablethat a5 to form ribozymes ofcreation artificial bond formation, alcohol oxidation, (Silverman Diels–Alder reaction 2009). the and The RNA as cleavage,such phosphorylation, ligation, capping, branching, and to peptide catalytic activity. theand fortested identifieddesired are zymes ribo cycles, protocol. of selected anumber selection selection After the greatly simplifying 60 catalytic activity (phenotype) become “linked” within individual droplets in a water-in-oil droplets individual in (phenotype) within activity catalytic become “linked” (Tawfikand 1998),and Griffiths (genotype) its ribozyme sequencethe the of which in IVC to use is strategies tag (Silverman limitation 2009). this around way One of getting RNA asubstrate a“capture” and the with labeled between formation of acovalent linkage multiple as such on properties generally based is turnover, selection since enzymatic asufficientlyratereaction fast with (Voytek aribozyme itandsince requires Joyce 2007). may that catalyzed be of reactions type the in method, itpower limited extremely is of this (VoytekRNA ligases Joyce and 2007; Wright Joyce and 1997).and Despite efficiency the to continuouslyfirst evolveRNA,they catalytic and were able to apply of twotypes it to (Wright more quickly Joyce and of times hundreds 1997). Joyce coworkers and were the evolution, continuous evolution, alternative toprotein-directed use is an occur can which However, manner. stepwise PACE to the analogous 3.2 for Section in system discussed evolution preformed a and are in of repeated selection rounds which occurs method, in activefolded way. acatalytically in was properly most RNA that previously of machine the RNA suggesting ligases, reported higher yieldproductthan a exhibited ribozyme artificial The reaction. uncatalyzed the thatRNA acceleratedligase the reaction were they able10 artificial to create an (Ikawa 2004). al. et arandom approach, was which into inserted scaffold region With this RNA an site reaction or motifs, modules the to formed construct that structural known centers. sites Ikawa catalytic used Guided al. et and modeling, bybinding molecular for therapeutic applications (Goto 2011; al. et Morimoto 2011). al. et acids nonproteinogenic and proteinogenic amino both containing peptides nonstandard platform to express of provide but RNA only molecules artificial also system an consisting catalytic of translation aprimitive not support existence hydroxy only the acids. Flexizymes and acids amino awide artificial array able of with acyl-tRNAsare to synthesize charged termed ribozymes, that “flexizymes,” synthetase-like aminoacyl-tRNA to create artificial Saito 2001), al. et Suga coworkers and evolution vitro repeated of cycles in used experiments 2003, al. et 2008a,b; al. et 2000; 2006; al. et Murakami Lee Niwa 2009; al. et Ohta 2007; al. et 2002; al. et (Bessho of studies Goto 2008a,b; al. et aseries In Kawakami tRNA synthetases. aminoacyl- called done of enzymes protein byinstead job is a family of tRNA; this terminus Joyce and other’s catalyze each (Lincoln ribozymes 2009). synthesis ligase RNA two which addition, evolution system in vitro in to create across-catalytic was used for “RNA its replication, the own world” supporting step required (Joyce theory 2007). In of catalyze RNA, polymerization akey catalyze the indeed RNA can can ribozyme natural 2007), Scott and provided (Robertson RNAof ligase an proof although no that known 2001; al. et (Johnston 2003, Bartel structure Lawrence and 2005), crystal the with together The reactions catalyzed by artificial ribozymes are many and range from RNA from and manyrange processing, are ribozymes by catalyzed artificial reactions The In the traditional in vitro selection process, there is no room to select for no to advancedroom is select process, there selection vitro in traditional the In approach selection development vitro traditional to ribozyme in The an to use is that delineate structures proteins, adoptRNA molecules,three-dimensional like defined capable 3 of the no aminoacylation RNA is the enzyme of there catalyzing nature, In ′ to 3 to The Nanobiotechnology Handbook Nanobiotechnology The ′ phosphodiester bond (Bartel 6 -fold over - ′

Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 Figure 3.5). This method, in combination with rational RNA engineering, yielded an RNA yielded an RNA rational engineering, with 3.5).combination Figure method, in This and amplificationthesorting fluorescence-activated extended cell of circle primers (FACS; extent by extension of of acombination the rolling primer emulsion,second detecting and by extension addition primer of primer/template triggering ribozymes, duplexes a in droplets to create the the within to occur transcription droplets,individual allowing in beads to magnetic attached of ribozymes library agenetic of encapsulating consists 2011). al. et (Wochner and bead-tagging, compartmentalized termed is method The RNA polymerase capable RNAs of an up to 95 nucleotidesto engineer of synthesizing emulsion. Recently, was developed strategy anovel approach on this selection based Enzymes Artificial (Modified from Wochner, A. et al.,et A. from Wochner, (Modified by FACSamplification. PCR by isolated are amplified and ribozymes active DNA. The the to (7) hybridized are probes fluorescent-labeled detection, (6)signal of a minicircle. DNAfacilitate To amplification circle by rolling is achieved amplification extension (5) Primer proceed. can extension primer and beads the from released are ribozymes (4) emulsion, beads. asecond In magnetic the to (cyan) attached template duplexes and are (black) primer and broken is (3) emulsion The hairpin. the to ligate subsequently that ribozymes producing emulsion, water-in-oil afirst within place takes circle). (2) (blue beads Transcription magnetic streptavidin-coated to attached are alibrary (red) genes from biotinylated and (green) oligonucleotides (1) ribozymes. Hairpin cial figure.) color for CD companion (See FIGURE 3.5 of goal acompletely to the closer us self-replicating ribozyme. bringing length, RNA template an from 2011), al. et (Wochner of its own half sequences polymerize can and active ribozyme enzymatically an was able new ribozyme to The synthesize ribozyme. polymerase greater activity,polymerase with fidelity,the parental than and generality 1 G 5 ene binding DNA circleannealing and rollingcircle amplificatio n 2 T ranscription and ligation Science 6 Compartmentalized bead-tagging method for the selection of artifi selection the for method bead-tagging Compartmentalized Fluorescent la , 332, 209, 2011.) 3 be Primer andtemplate ling binding 7 FA and amplification CS, gene 4 re Primer extension cover y, 61 - Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 3.9 (Silverman 2009). counterparts natural their perform are ablethey to outenzymes, with competeprotein cannot ribozymes although artificial values of up to 10 maximum abzymes, with with observed 62 (d) (d) (a) foldamers. of various backbones chemical of the Comparison FIGURE 3.6 ofsequence enantiomers, the rapidly. foldamers, as progressing known is folding polymers, of biomimetic types applications. on these Research future in proteins 3.6) (Figure Alternative side may and backbones prove chains to have advantages over folds functions. and capable of also polymers are three-dimensional of protein-like types out nucleic of and protein acids, its to nanomachines build other chosen has Although nature The rate enhancements achieved with artificial ribozymes are are comparable the ones with ribozymes artificial achieved with rateThe enhancements Nature has elected to use only only to use elected has Nature β 2 -peptide, and (e) and -peptide, Other Nonprotein Artificial Enzymes Nonprotein Artificial Other d -amino acids will fold into a mirror-image protein with activity against against activity with fold protein amirror-image into acids will -amino β d 3 -amino acids. In principle, for each natural protein, a corresponding principle, acids. protein, In acorresponding for natural -amino each -peptide. (e) (d) (c) (b) (a) N H N N N N H H H R 1 R R R 1 1 1 l -amino acids at the expense of their mirror-image mirror-image of their acids at expense the -amino O O O R 1 OO O N N N H H R 2 N N R R 2 2 O O O R 2 N N N R R H H 2 3 O R R 3 3 O O O N N The Nanobiotechnology Handbook Nanobiotechnology The −10 l R N N N H H R -peptide, (b)-peptide, 4 3 s −1 (Suga 1998). al. et However, d -peptide, (c)-peptide, peptoid, - Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 cover aconsiderable space. of amount However, chemical includethat proteins artificial natural life. with to biological prevent interfering from systems synthetic escaped mechanism safety 2007), (Forster Church diastereomers and and a of or as enantiomers synthesis for the 2011). al. et Granillo of entirely composed proteins Full DeGradoand 2006) incorporated into and candidates. drug promising substrates. mirror-image Enzymes Artificial which adopts the noncovalent interaction-based specificity strategy that is so successful so successful is that adopts which noncovalentstrategy the specificity interaction-based aself-assembling to build Computational 2009) al. (Butterfoss et designs. was recently design used to facilitate future extended with for being use are design and to fit prediction parameters, computationaldeveloped methods for structure protein which knowledge against base vivo degradation. structural Despite of lack anatural the to in peptides, greatly reduced with susceptibility of antimicrobial foldthe function and (Qiupeptoids 2006) al. to synthesize. et than butstructure more are difficult (Appella 1997) al. et structure cooperative and secondary noncovalent helical quaternary ( bonded group is to the amino bonded group is to the amino the acids, which in amino (Maayan peptoid of 2009). al. et the used handedness depended on the which catalysis, enantioselective peptoid in resulted molecule catalyst to astructured small atemplate. interface as aphase achiral did not an and require Fusing side chains charged of positively of hydrophobicdriven pairing negatively burial by and and the side chains at a1:1 ratio, spontaneously a2.7 36-mers formed two the aqueous solution in sheets 2010). al. (Nam et crystalline mixed When two-dimensional form study, another In structure. peptoid two target polymers the were to reported assumed proteins) in peptoid were of only the zinc to bind positioned zinc to bind used histidines and cysteines the by al. et (inspired 2008). (Lee imidazole sideandchains Thiol affinity nanomolar with selectively that zinc was bound designed example, bundle atwo-helix peptoids. by designed demonstrated one sequence-levelwith In been has design function of polymer mRNA. Programming amplificationpeptoids isolation and encoding via of developed 2008b) al. et been for enzymatic could(Kawakami that also enable has selections system ribosomal engineered An amines. available of commercially primary thousands (Burkoth 2003) al. et submonomer method allows incorporation of that synthetic any of byadvantage aconvenient polymerized be economical and can of they peptoids that is protein. same acids the into amino incorporation of multiple the allow can method noncanonical stop codon, amber the this on suppression based strategies earlier acids. of Unlike amino noncanonical containing proteins artificial nucleotide quadruplet to codons synthesize recognized that ribosome 2011). al. et 2010) al. et (Neumann project modified a used interesting particularly One extensively been reviewed has and (e.g., acids proteins into amino noncanonical Antonczak Extensive work doneincorporation been to enable global orof has site-specific the even an wider perform range of acids could functions. potentially amino noncanonical β 2 Natural proteins are composed of 20 canonical amino acids, among them which amino of composed 20 are canonical proteins Natural Enzyme-like catalysts are also emerging from the field the chemistry, from of supramolecular emerging also catalysts are Enzyme-like Both of foldamers the class is promising Another Peptoids, Akey peptide analogs. protease-resistant or glycines, achiral, N-substituted are -peptides) or the β -peptides (Porter 2002) al. et peptoids and (Chongsiriwatana 2008) al. et mimic can β carbon ( carbon d -peptides are resistant to degradation by proteases, making them them to degradation resistant by-peptides are proteases, making β -peptide hexameric bundle (Korendovych bundle -peptide hexameric 2010). al. et d β -amino acids have computationally been -amino modeled (Nanda 3 -peptides). -peptides). β carbon. The side chain can branch off of the of off the branch can side The carbon. chain β β -peptides (Shandler 2010) al. et peptoids and 3 -peptide foldamers have shown to adopt been l -peptides to improve (Rodriguez- stability β -peptides. As opposed to standard to-peptides. standard opposed As d -amino acids could be very useful acids could very be useful -amino nm-thick bilayer.nm-thick Assembly was α carbon, in in carbon, β amino acids, the amino α carbon carbon 63 α

Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 nucleic favor acids in at of suited task for best whatever hand. the is scaffold chemical and to abandon free be protein and will we enzymes, artificial of nanobiotechnological rapidly.closing gap no longer narrows, we dependent be will this As on nature’s examples center clamp. of the the through was threaded active polymer when chain the catalyst The was only chain. growing the secured that clamp amolecular as acted other active catalytic site the the and contained ring heterodimer. cyclodextrin One cyclodextrin effectively polymerize for biological catalysts. Recently, of DNA polymerase was acatalyst shown to reminiscent 64 point withsome amountofthedesired activity. Ifthis activityhasnotbeenfound innature, starting pointsforoptimization bydirected evolution, whichgenerallyrequires astarting or designedcomputationally. Poorcatalysts,whilethemselves notuseful,canserveas especially rare. Thesesequencescanbeselectedfrom relatively smallconstrainedlibraries been discovered by millenniaofevolution,sequencesthatencodepoor catalystsare not that encode highly efficient enzymesare extremely rare, andtothis point haveonly review provides encouragement for the future. It seems possible that whilesequences undesignable. fundamentally and may provechaotic Indeed, efficient as be enzymes to tuned so complexbe to finely and future. the to forforeseeable design difficult extremely remain will subtle mechanisms active Despite conformation. an into rapid improvements computational in power, such favorstyle in the protein “induced of the changes fit” substrate in model, binding which 2007a,b). al. et “lock-and-key” out Indeed, the fallen of catalysis has model of enzyme 2006; Benkovic and Henzler-Wildman toprotein accelerate (Hammes-Schiffer reactions the within transitions motion and structural harness that mechanisms subtle catalytic may byalone. have evolving advantagestate Proteins stabilization taken softness of their floppy too are catalysistransition proteins effective by perform to like bionanomachines TSAs. to bind out It raised “soft” that are may which turn antibodies, to catalytic activity have that similar yielded state enzymes has transition the to stabilize theozymes designing work. computational do the of that strategy their proteins by which It interesting is subtle ofmechanisms the by poor understanding our but constrained we remain relaxing, powerworked processor and slowly computational are in out. algorithms Limitations developmentthe nonprotein nanomachines. of biologically inspired applydirectly to will proteins artificial and study the ofknowledge from natural gained The progress. and understanding to our instrumental be will nanomachines functional well, examples study as of close of natural nanotechnology In architecture. design, and product avaluablecomplex engineering, been has in strategy problems. Biomimicry ofprocess evolution, found remarkably has clever efficient and nature ways solving of the Through industries. health would and revolutionizetransformation chemical the 2011).chemical al. et any perform to desired enzymes to create artificial ability The applications, vivo 2010) in (Keasling both medicinal and (Zhang of vitro industrial in and awide roles variety in playing catalysts increasing and traditional replacing are Enzymes 3.10 These studies show that the gap between protein science and nanoscale chemistry is is chemistry nanoscale and science protein show gap studies the between that These The successoftheprotein design,evolution,andselectionstudieshighlightedinthis rapidly,is progressing design enzyme be to butproblemsremain daunting Artificial Conclusions Perspectives and δ -valerolactone 2011). al. et (Takashima of catalyst a The consisted The Nanobiotechnology Handbook Nanobiotechnology The Downloaded By: 10.3.98.104 At: 17:55 26 Sep 2021; For: 9781439838709, chapter3, 10.1201/b12935-5 The eliminationofunnecessarycomplexityisacentraltenetsyntheticbiology(Endy (or perhaps accumulated) bynature maynotbenecessaryforhuman-definedtasks. to transform the formerinto the latter. However, theincredible complexity developed (Fisher etal.2011) wouldsuggestthatatremendous amountofevolutionwouldbeneeded structures ofa102-residue helicalbundleandthecitratesynthaseitfunctionallyreplaced efficiency approaching thoseofnaturalproteins. Comparisonofthethree-dimensional poor catalyststotheextremely rare sequencethatencodesahighlyoptimizedenzymewith evolutionary pathways through sequence space exist connecting these relatively abundant synthetic enzymeswithminimalactivitycouldbeveryvaluable.Thequestioniswhether Enzymes Artificial Chen, Z.andZhao,H.(2005). Rapidcreation ofanovel protein functionbyin vitro coevolution. Chen, I.,Dorr, B.M., andLiu,D.R.(2011). A generalstrategy fortheevolutionofbond-forming Butterfoss, G.L.,Renfrew, P.D., Kuhlman,B.,Kirshenbaum,K.,andBonneau,R. (2009). A preliminary Burkoth, T.S., Fafarman, A.T., Charych,D.H.,Connolly, M.D.,andZuckermann,R.N.(2003). Boyer, P.D. (1997).The ATP synthase—A splendidmolecularmachine. Bolon, D.N.andMayo,S.L.(2001).Enzyme-likeproteins bycomputationaldesign. Bessho, Y., Hodgson,D.R.,andSuga,H.(2002). A tRNA aminoacylationsystemfornon-natural Bertini, I.,Gray, H.B.,Stiefel,E.I.,and Valentine, J.S. (2007). Benkovic, S.J. and Hammes-Schiffer, S. (2003). A perspective on enzyme catalysis. Belogurov, A., Jr., Kozyr, A., Ponomarenko, N.,andGabibov, A. (2009).Catalyticantibodies:Balanc­ Bartel, D.P. andSzostak,J.W. (1993).Isolationofnewribozymesfrom alarge poolofrandom Appella, D.H.,Christianson,L.A.,Klein,D.A.,Powell,D.R.,Huang,X.,Barchi, J.J.,andGellman,S.H. Antonczak, A.K., Morris,J.,andTippmann, E.M.(2011). Advances inthemechanismand Anfinsen, C.B.,Haber, E.,Sela,M.,andWhite, F.H. 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