Artificial Enzymes - The Nanobiotechnology - page 57

DFtet,. to. accommodate. the. substrate,. they. were. able. to. engineer. a. variant. capable. of.
catalyzing.the.oxidation.of.4-aminophenol.in.the.presence.of.atmospheric.oxygen.with.
a.10 3 -fold.rate.enhancement.(Kaplan.and.DeGrado.2004)..However,.this.redesign,.which.
included.the.mutation.of.an.Ala.and.a.Leu.to.two.Gly.residues.in.two.of.the.four.chains,.
signiicantly. destabilized. the. protein.. More. recently,. the. interhelical. turn. of. DF1. was.
modiied.with.the.goal.of.overcoming.the.conformational.destabilization.introduced.by.
the. Gly. mutations. (Faiella. et. al.. 2009).. This. new. design,. which. also. incorporated. Gly.
mutations.in.the.active.site.pocket,.was.termed.DF3.and.was.able.to.catalyze.the.oxidation.
of.4-aminophenol.and.3,5-ditert-butyl-catechol..The.new.design.also.exhibited.improved.
thermodynamic. stability. with. respect. to. previous. variants. and. remained. active. for. at.
least.50.cycles.(Faiella.et.al..2009).
3.7 De Novo Design of Enzymes without Metal Cofactors
While. pragmatic. evolution-based. methods. have. been. very. successful. in. solving. limited.
classes.of.problems,.the.ability.to.design.a.sequence.that.will.fold.and.function.as.predicted.
remains.the.Holy.Grail..This.goal.provides.the.ultimate.test.of.our.understanding.of.protein.
folding.and.enzymatic.mechanism,.and.of.our.ability.to.control.physics.and.chemistry.at.
the.nanoscale.
Thanks. to. the. continued. increases. in. computer. speed,. computation. has. emerged. as. a.
promising.method.for.taming.the.complexity.of.enzyme.design..Thus.far,.computational.
studies.have.focused.on.designing.new.active.sites.for.incorporation.into.existing.protein.
scaffolds.. Over. a. decade. ago,. an. active. site. was. designed. into. a. thioredoxin. scaffold. to.
create.a.new.enzyme.that.hydrolyzed.p-nitrophenyl.acetate.(PNPA;.Bolon.and.Mayo.2001)..
Using.a.strategy.similar.to.that.underlying.catalytic.antibody.generation,.the.active.site.was.
designed.to.stabilize.a.high-energy.intermediate.along.the.reaction.pathway..A.designed.
enzyme. containing. only. three. mutations. relative. to. the. wild. type. was. able. to. catalyze.
PNPA.hydrolysis.with.kinetics.similar.to.those.of.early.abzymes.
Computational.redesign.of.a.substrate-contacting.loop.changed.the.speciicity.of.human.
guanine.deaminase.2.5.×.10 6 -fold.in.favor.of.a.target.substrate,.ammelide.(Murphy.et.al..
2009)..The.new.loop,.which.at.four.residues.in.length.was.two.residues.shorter.than.the.
original. loop,. placed. an. asparagine. residue. in. position. to. form. hydrogen. bonds. with. a.
docked. ammelide. molecule.. A. crystal. structure. of. the. designed. enzyme. revealed. that.
the.coniguration.of.the.designed.loop.matched.the.design.to.a.alpha-carbon.root.mean.
square. deviation. (Cα-RMSD). of. 1.Å.. Point. mutants. conirmed. that. correct. placement. of.
the.designed.asparagine.residue.was.important.for.activity..However,.the.activity.of.the.
designed.enzyme.with.ammelide.was.seven.orders.of.magnitude.lower.than.that.of.the.
wild-type.enzyme.with.guanine.
In.a.series.of.recent.breakthrough.reports,.David.Baker.and.his.colleagues.have.described.
a.novel.protocol.for.the.creation.of.artiicial.enzymes..In.contrast.to.approaches.in.which.
the. scaffold. is. chosen. irst,. the. irst. step. of. the. Baker. protocol. (Figure. 3.4). is. to. design.
and. computationally. model. disembodied. idealized. active. sites. for. the. target. reaction..
Like. abzymes,. these. theoretical. enzymes,. or. “theozymes,”. are. designed. to. stabilize.
the. transition. state. of. the. reaction.. Quantum-mechanical. calculations. are. then. used. to.
identify.the.most.promising.designs..Once.a.large.collection.of.candidate.theozymes.has.
been.generated,.the.RosettaMatch.program.attempts.to.graft.each.of.these.constellations.