Artificial Enzymes - The Nanobiotechnology - page 51

infect.fresh.bacteria.and.replicate.fast.enough.to.avoid.being.washed.out..The.phage.life.
cycle.is.on.the.order.of.10.min,.faster.than.that.of.the.bacteria.and.shorter.than.the.residence.
time.within.the.lagoon..The.phage.are.modiied.to.contain.a.copy.of.the.gene.to.be.evolved,.
which. is. expressed. upon. infection. of. a. bacterial. host.. The. desired. enzymatic. activity. is.
linked.to.the.production.of.pIII,.a.phage.protein.required.for.infectivity..Therefore,.phage.
harboring. genes. that. encode. active. proteins. replicate. within. cells. to. produce. infectious.
phage..Genes.that.encode.proteins.lacking.the.desired.activity.also.produce.new.phage,.
but.these.are.unable.to.infect.new.host.cells.and.harmlessly.wash.out.of.the.lagoon..The.
high.natural.error.rate.of.phage.replication.generates.a.diverse.set.of.random.mutations.
within.the.target.gene.and.can.be.optionally.enhanced.by.a.mutagenesis.plasmid..Esvelt.
and.coworkers.demonstrated.the.power.of.PACE.by.evolving.new.versions.of.the.T7.RNA.
polymerase.that.recognize.the.T3.promoter.and.that.initiate.with.ATP.or.CTP.rather.than.
GTP.. PACE. enabled. up. to. 200. rounds. of. evolution. to. occur. over. 8. days. with. no. human.
intervention.. The. researchers. were. able. to. follow. the. mutational. paths. taken. in. initially.
identical.parallel.runs:.in.the.T3.promoter.experiment,.two.lagoons.accumulated.different.
mutations. before. converging. upon. the. same. optimal. set.. While. PACE. is. limited. to. the.
evolution. of. enzymatic. activities. that. can. be. linked. to. pIII. production,. the. beneits. of.
continuous.evolution.will.motivate.imaginative.protein.engineers.to.think.of.clever.ways.
to.establish.such.a.linkage.for.a.wide.variety.of.enzymes.
Directed. evolution. has. the. beneit. of. requiring. no. information. about. the. structure. or.
mechanism.of.the.enzyme..The.same.“blind.watchmaker”.that.built.the.parent.enzyme.
adapts.it.to.the.demands.of.its.new.environment..However,.because.functional.proteins.
are.islands.in.the.vastness.of.sequence.space,.it.is.unwise.to.stray.too.far.from.the.parental.
sequence. by. introducing. too. many. mutations. at. once.. Directed. evolution. is,. therefore,.
better. suited. for. small. optimizing. tweaks. than. it. is. for. introducing. large. changes. like.
entirely.new.folds.or.functions..In.a.few.cases,.however,.directed.evolution.has.succeeded.
in. generating. enzymes. with. activities. not. present. in. the. parent. enzyme.. One. effective.
strategy. has. been. to. use. multistep. evolutionary. paths. in. which. one. or. more. bridging.
substrates.span.the.structural.gap.between.a.wild-type.substrate.and.a.desired.substrate..
For. example,. in. the. PACE. study,. the. wild-type. T7. RNA. polymerase. showed. no. activity.
with.the.T3.promoter,.and.selection.for.activity.on.that.promoter.did.not.support.phage.
propagation.. Selection. for. activity. on. a. hybrid. promoter. consisting. of. the. T3. promoter.
sequence.with.the.T7.promoter.base.at.the.−11.position.followed.by.selection.on.the.full.T3.
promoter.succeeded.in.generating.the.desired.mutant..Similarly,.the.steroids.testosterone.
and. progesterone. were. used. to. bridge. the. structural. gap. between. the. natural. substrate.
of.human.estrogen.receptor.α.ligand-binding.domain,.17β-estradiol,.and.the.inal.target.
substrate.corticosterone.(Chen.and.Zhao.2005)..However,.while.this.strategy.can.be.effective.
for.altering.the.substrate.speciicity.of.a.particular.type.of.activity.when.clear.structural.
intermediaries. between. the. natural. and. target. substrates. exist,. it. may. be. impossible. to.
extend.it.to.the.creation.of.enzymes.that.catalyze.new.reactions.
Another. approach. to. generating. new. activity. combined. rational. design. and. directed.
evolution. to. introduce. β-lactamase. activity. into. a. glyoxalase. II. (GlyII;. αβ/βα). metallo-
hydrolase.(Park.et.al..2006)..The.C-terminal.domain.of.the.parent.enzyme.was.removed.to.
relieve.steric.constraints,.and.substrate-.and.metal.cofactor-binding.loops.derived.from.a.
sequence.alignment.of.metallo.β-lactamase.(MBL).enzymes.were.inserted.into.the.scaffold.
along.with.targeted.mutations.that.introduced.catalytic.residues..This.rationally.designed.
scaffold. served. as. the. parent. for. random. directed. evolution. by. error-prone. polymerase.
chain. reaction. (PCR). and. DNA. shufling. (Stemmer. 1994),. resulting. in. the. isolation. of. a.
mutant. that. could. support. E. coli . growth. in. the. presence. of. a. 1.0.μg/mL. concentration. of.