Part One Amino Acids as Building Blocks

82 Pages·2010·5.86 MB·English

Part One
Amino Acids as Building Blocks
AminoAcids,PeptidesandProteinsinOrganicChemistry.
Vol.3–BuildingBlocks,CatalysisandCouplingChemistry.EditedbyAndrewB.Hughes
Copyright(cid:1)2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:978-3-527-32102-5

j
3
1
Amino Acid Biosynthesis
EmilyJ.ParkerandAndrewJ.Pratt
1.1
Introduction
Theribosomalsynthesisofproteinsutilizesafamilyof20a-aminoacidsthatare
universally coded by the translation machinery; in addition, two further a-amino
acids, selenocysteine and pyrrolysine, are now believed to be incorporated into
proteinsviaribosomalsynthesisinsomeorganisms.Morethan300otheraminoacid
residueshavebeenidentifiedinproteins,butmostareofrestricteddistributionand
produced via post-translational modification of the ubiquitous protein amino
acids[1].Theribosomallyencodeda-aminoacidsdescribedhereultimatelyderive
from a-keto acids by a process corresponding to reductive amination. The most
importantbiosyntheticdistinctionrelatestowhetherappropriatecarbonskeletons
arepre-existinginbasicmetabolismorwhethertheyhavetobesynthesizeddenovo
andthisdivisionunderpinsthestructureofthischapter.
Thereareasmallnumberofa-ketoacidsubiquitouslyfoundincoremetabolism,
notably pyruvate (and a related 3-phosphoglycerate derivative from glycolysis),
together with two components of the tricarboxylic acid cycle (TCA), oxaloacetate
and a-ketoglutarate (a-KG). These building blocks ultimately provide the carbon
skeletonsforunbrancheda-aminoacidsofthree,four,andfivecarbons,respectively.
a-Amino acids with shorter (glycine) or longer (lysine and pyrrolysine) straight
chainsaremadebyalternativepathwaysdependingontheavailablerawmaterials.
The strategic challenge for the biosynthesis of most straight-chain amino acids
centersaroundtwoissues:howisthea-aminofunctionintroducedintothecarbon
skeleton and what functional group manipulations are required to generate the
diversityofside-chainfunctionalityrequiredfortheproteinfunction?
Thecorefamilyofstraight-chainaminoacidsdoesnotprovideallthefunctionality
required for proteins. a-Amino acids with branched side-chains are used for two
purposes;theprimaryneedisrelatedtoproteinstructuralissues.Proteinsfoldinto
well-defined three-dimensional shapes by virtue of their amphipathic nature: a
significant fraction of the amino acid side-chains are of low polarity and the
hydrophobic effect drives the formation of ordered structures in which these
side-chains are buried away from water. In contrast to the straight-chain amino
AminoAcids,PeptidesandProteinsinOrganicChemistry.
Vol.3–BuildingBlocks,CatalysisandCouplingChemistry.EditedbyAndrewB.Hughes
Copyright(cid:1)2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:978-3-527-32102-5
j
4 1 AminoAcidBiosynthesis
acids,thehydrophobicresidueshavelargenonpolarsurfaceareasbyvirtueoftheir
branched hydrocarbon side-chains. The other role of branched amino acids is to
providetwousefulfunctionalgroups:animidazole(histidine)andaphenol(tyrosine)
thatexploitaromaticfunctionalgroupchemistry.
This chapter provides an overview of amino acid biosynthesis from a chemical
perspective and focuses on recent developments in the field. It highlights a few
overarchingthemes,includingthefollowing:
i) Thechemicallogicofthebiosyntheticpathwaysthatunderpinaminoacidbio-
synthesis.Thischemicalfoundationiscriticalbecauseoftheevolutionarymecha-
nismsthathaveshapedthesepathways.Inparticular,thewayinwhichgene
duplicationandfunctionaldivergence(viamutationandselection)cangenerate
newsubstratespecificityandenzymeactivitiesfromexistingcatalysts[2].
ii) The contemporary use of modern multidisciplinary methodology, including
chemistry, enzymology, and genomics, to characterize new biosynthetic
pathways.
iii) Potential practical implications of understanding the diverse metabolism of
aminoacidbiosynthesis,especiallymedicinalandagrichemicalapplications.
iv) Thehigher-levelmoleculararchitectures thatcontrolthefateofmetabolites,
especially the channeling of metabolites between active sites for efficient
utilizationofreactiveintermediates.
Box1.1: NitrogenandRedoxinAminoAcidBiosynthesis
Ammonia is toxic and the levels of ammonia available for the biosynthesis of
aminoacidsinmostbiochemicalsituationsislow.Therearealimitednumberof
entrypointsofammoniaintoaminoacidbiosynthesis,notablyrelatedtogluta-
mate and glutamine. Once incorporated into key amino acids, nitrogen is
transferredbetweenmetaboliteseitherdirectlyorviainsituliberationofammonia
byamultifunctionalcomplexincorporatingthetargetbiosyntheticenzyme.The
mainsourceofinsitugeneratedammoniaforbiosynthesisisthehydrolysisof
glutamine by glutaminases. De novo biosynthesis of amino acids, like element
fixationpathwaysingeneral,isprimarilyreductiveinnature.Thismayreflectthe
originsofthesepathwaysinananaerobicworldmorethan3billionyearsago.
Box1.2: TheStudyofBiosyntheticEnzymesandPathways
Thesourceofanenzymeforbiochemicalstudyhasimportantimplications.Most
coremetabolismhasbeenelaboratedbystudyingasmallnumberoforganisms
that werechosenforavarietyofreasons,includingavailability,easeofmanip-
ulation,ethicalconcerns,scientificcharacterization,andsoon.Theseexemplar
organismsincludethebacteriumEscherichiacoli,theyeastSaccharomycescerevi-
siae,theplantArabidopsisthaliana,andtheratasatypicalmammal.Muchofthe
detailedcharacterizationofaminoacidbiosynthesiscommencedwithstudieson
theseorganisms.Withtheriseofgeneticengineeringtechniques,biosynthetic
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1.2 GlutamateandGlutamine:GatewaystoAminoAcidBiosynthesis 5
enzymesfromawidevarietyofsourcesareavailableforscientificinvestigation,
andtherehasbeenincreasingemphasisonworkingwithenzymesandpathways
fromalternativeorganisms.
Metabolicdiversityisgreatestamongprokaryotes.Onefundamentalchangein
the underlying microbiology that has affected our understanding of pathway
diversityhasbeentheappreciationofthedeepbiochemicaldistinctionsbetween
what are now recognized to be two fundamental domains of prokaryotes:
eubacteriaandArchaea[3].Theformerbacteriaincludethosewellknowntobe
associatedwithdiseaseandfermentationprocesses;whilethelatterincludemany
methanogensandextremophiles(prokaryotesthatgrowinextremeconditions,
suchashyperthermophilesthatgrowattemperaturesabove60(cid:2)Corhalophiles
thatgrowinhighionicstrengthenvironments).Bioinformaticsapproachesare
complementing conventional enzymological studies in identifying and charac-
terizinginterestingalternativebiosyntheticpathways[4].Thegreaterunderstand-
ingofmicrobialandbiosyntheticdiversityispresentingexcitingopportunitiesfor
noveldiscoveriesinbiosynthesis.
Muchofthefocusofbiosyntheticenzymologynowfocusesonenzymesfrom
pathogens and hyperthermophiles. The focus on the study of enzymes from
pathogensispredicatedonthepossibilitythatinhibitorsofsuchenzymesmaybe
usefulaspesticidesandtherapeuticagents.Sincehumanshaveaccesstomany
aminoacidsintheirfood, theyhavelosttheabilitytomake“dietaryessential”
aminoacidsthattypicallyrequireextendeddedicatedbiosyntheticpathways[5].
Thebiosyntheticenzymesofthecorrespondingpathwaysareessentialformany
pathogens and plants, but not for humans; hence, selective inhibitors of these
biosyntheticenzymesarepotentiallynontoxictohumans,buttoxictoundesirable
organisms. Enzymes from hyperthermophilic organisms, produced by genetic
engineering, are scrutinized mainly because of their ease of structural charac-
terization. These enzymes retain their native structures at temperatures that
denature most other proteins, including those of the host organism. These
proteinsareofhighthermalstabilityandsimpleheattreatmentcanbeusedto
effecthighlevelsofpurificationofthedesiredprotein.
1.2
GlutamateandGlutamine:GatewaystoAminoAcidBiosynthesis
Glutamateandthecorrespondingamidederivative,glutamine,arecriticalmetabo-
lites in amino acid metabolism. The biochemistry of these two amino acids also
illustratesthedistinctchemistryassociatedwiththea-aminoandside-chainfunc-
tionalgroups,eachofwhichisexploitedinthebiosynthesisofotheraminoacids.
These amino acids derive from ammonia and a-KG. Glutamate dehydrogenase
(GDH) interconverts a-KG and glutamate (Figure 1.1) [6]. Although glutamate is
formedinthiswaybyreductiveamination,thisenzymeisgenerallynotdedicatedto
biosynthesis;thereversereaction,anoxidativedeaminationtoregeneratea-KG,is
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6 1 AminoAcidBiosynthesis
CO2 GDH CO2
NH3 + NAD(P)H + + H2O + NAD(P)+
O CO H N CO
2 3 2
α-ketoglutarate glutamate
Figure1.1 Interconversionofa-KGandglutamatecatalyzedbyGDH.
ATP ADP NH3 HOPO32
CO CO PO 2 CONH
2 2 3 2
H N CO GS H N CO GS H N CO
3 2 3 2 3 2
Figure1.2 ConversionofglutamatetoglutaminecatalyzedbyGS.
oftenanimportantinvivoroleforthisenzyme[7].Thisdeaminationchemistrymight
beafactorintherelativelyweakbindingofammonia(e.g.,K (NH )is3mMforthe
M 3
E.colienzyme–abovenormalenvironmentalconcentrations).Inmanyorganisms
there is an additional enzyme, glutamate synthase (GOGAT), dedicated to the
biosynthesis of glutamate [8]. GOGAT utilizes ammonia generated in situ by
the hydrolysis of glutamine and this enzyme will be described after a discussion
ofthebiosynthesisofglutamine.
The conversion of glutamate to glutamine, catalyzed by glutamine synthetase
(GS),requirestheactivationoftheside-chaincarboxylateasanacylphosphate,prior
to nucleophilic substitution of the resulting good leaving group by ammonia
(Figure 1.2). The use of ATP, to produce c-glutamyl phosphate, assists both
the kinetics and the thermodynamics of amide formation: by producing a more
reactivecarboxylicacidderivativeandoverturningtheintrinsicallyfavorablenature
ofamidehydrolysisinwater.
GSfromentericbacteria,suchasE.coliandSalmonellatyphimurium,isanexemplar
ofanaminoacidbiosyntheticenzyme;theoverallreactionitcatalysesiseffectively
irreversibleinvivo (K¼1200). Beingdedicatedtobiosynthesis,ithas evolvedtight
bindingofammonia(K (NH )<200mM)whichallowsefficientsynthesisofgluta-
M 3
mineunderthelowammoniaconditions(muchlessthan1mM)foundinvivo.Itsin
vivoroleasanentrypointforthebiosynthesisofawiderangeofnitrogenmetabolitesis
eloquentlycommunicatedbytheextensivefeedbackregulationofthisenzymebya
range of nitrogen-containing metabolites, including glycine, serine, alanine, and
histidine[9–12].Glutamineistheprimarystoreofammoniainmanycells;theside-
chainamideischemicallyunreactive,butitsfavorablehydrolysiscanbecatalyzedon
demandbyglutamineamidotransferase(GAT)enzymes[13].
1.2.1
CaseStudy:GOGAT:GATsandMultifunctionalEnzymesinAminoAcidBiosynthesis
IncontrasttoGDH,GOGATisadedicatedbiosyntheticenzyme.Itistheprimary
source of glutamate in plants, eubacteria and lower animals [14, 15]. These iron–
sulfurflavoproteinscarryoutthereductiveaminationofa-KGtoglutamateviaafive-
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1.2 GlutamateandGlutamine:GatewaystoAminoAcidBiosynthesis 7
GOGAT
NH
Glustuabmuinniatse H Cys21Enz CO-SEnz + H2O CO2
S H3N CO2 H3N CO2
NH
2
O + H
H N CO
3 2
NH3 FMNH2 FMN
CO
2
+ H O
2
H N CO
3 2
GOGAT
CO
Synthase 2
subunit
O CO
2
Figure1.3 ThebiosynthesisofglutamatemediatedbyGOGAT.
stepprocessthatutilizestheinsituhydrolysisofglutamineasthesourceofammonia
forthisreaction(Figure1.3)[16].
Aswithmanyreductivebiosyntheticenzymes,therearevariantsoftheenzyme
adapted to different electron sources; for example, both ferredoxin- and nicotin-
amide-dependent enzymes are known, and examples of both of these classes of
GOGAT have been studied in detail [15]. They reveal many of the key features of
metabolite channeling observed in biosynthetic enzymes utilizing glutamine as a
nitrogendonor.
The NADPH-dependent GOGAT from Azospirillum brasilense is an
a,b-heterodimer [17]. The b-subunit supplies the electrons for the reductive
amination process: NADPH reduces FADand the electrons are passed, in turn,
toa3Fe–4Scenteronthea-subunitandthenontotheFMNcofactorattheactive
site for glutamate formation. The a-subunit consists of four domains. The N-
terminal domain is a type II GAT, in which the N-terminal cysteine attacks
glutaminereleasingammoniaandgeneratinganenzyme-boundthioester,which
is subsequently hydrolyzed (Figure 1.3) (type I GATs, the other variant, utilize a
combinationofaninternalcysteineandahistidineascatalyticresidues[18]).When
aclassIIGATisactive,aconservedQ-loopclosesovertheactivesiteandprevents
releaseofammoniatothesolution;insteadthenascentammoniatravelsthrougha
hydrophilicinternaltunnelapproximately30Åinlengthtothethirddomainwhich
isa(ba) barrelcontainingthe3Fe–4SclusterandtheFMNactivesite.Thelatter
8
sitebindsthesubstratea-KGandcarriesoutthesynthesisofglutamate,presum-
ably via reduction of an a-iminoglutarate intermediate. There is a gating mech-
anism for synchronization of GAT and reductive amination active sites: the
glutaminase activity is dependent on the binding of both a-KG and reduced
cofactoratthesecondsite(Figure1.4)[19].
The ferredoxin-dependent GOGAT from the cyanobacterium, Synechocystis sp.
PCC6803,hasasimilarstructuretotheA.brasilenseenzyme,possessingatypeII
GATdomain and a synthase site linked by a 30-Å tunnel, which is gated in an
analogousway[21].TheGATdomainexistsinaninactiveconformation,whichcan
bind glutamine but not hydrolyze it. This is converted to the active conformation
on binding of a-KG and reduced cofactor, FMNH , to the synthase site; this
2
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8 1 AminoAcidBiosynthesis
Figure1.4 StructureofGOGATshowingtheinternaltunnelforammoniatransferbetweenthe
GAT(gold)andsynthase(blue)activesites.(Picturetakenfrom[20].)
conformationalswitchalsoservestoopentheentrypointtotheammoniatunnel.A
conservedglutamateresidue(Glu1013intheSynechocystisenzyme),presentatthe
tunnel constriction, has been shown to be the key residue controlling the cross-
regulationmechanism.ThisglutamateinteractswiththeN-terminalaminogroupof
theprotein,whichistheactive-sitebaseoftheglutaminase,aswellasaffectingthe
geometryofthetunnelentrypoint.Mutationofthisresiduetoaspartate,asparagine,
oralanineaffectedglutaminaseactivityandthesensitivityofglutaminaseactionto
thebindingofa-KGatthesynthasesite[22].
GOGATexemplifies our growing awareness of details of glutamine-dependent
enzymes, in particular, and biosynthetic pathways, in general. By exploiting the
higher-level organization of multifunctional enzyme systems, metabolites can be
channeledtothenextenzymeofapathway;therebycontrollingtheirfate.Together
with the potential for subtle levels of regulation, this organization ensures the
efficientuseofbiosyntheticintermediates[20].
1.3
OtherAminoAcidsfromUbiquitousMetabolites:PyridoxalPhosphate-Dependent
RoutestoAspartate,Alanine,andGlycine
1.3.1
PyridoxalPhosphate:ACriticalCofactorofAminoAcidMetabolism
Onceglutamateisavailable,thea-aminofunctioncanbetransferredtoothera-keto
acids via amino acid aminotransferase enzymes (Figure 1.5) [23]. This family of
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1.3 OtherAminoAcidsfromUbiquitousMetabolites:PyridoxalPhosphate-DependentRoutes 9
CO CO
2 2
H N CO O CO
3 2 2
Enz-PLP Enz-PMP
R R
H N CO O CO
3 2 2
Figure1.5 Overallinterconversionmediatedbymostaminotransferaseenzymes(e.g.,for
AATasesR¼CHCO(cid:3)).
2 2
enzymes exploit the catalytic versatility of the cofactor pyridoxal 50-phosphate
(pyridoxal phosphate PLP), one of the active forms of vitamin B , which is inter-
6
converted with pyridoxamine phosphate (PMP) during the overall transformation
[24,25].
ThealdehydeofPLPreadilyformsSchiffbaseswithaminesandthiscofactoris
generallytetheredtotheactivesiteofenzymesviaalinktoalysineside-chain.Amino
acidsubstratesbindtothecofactorbySchiffbaseexchangewiththeenzymelysine,
whichistherebyliberatedasapotentialactive-sitebase.Thecriticalfeatureexploited
inaminoacidmetabolismistheabilityofPLPtoactasanelectronsink,stabilizing
a
negativechargebuildupatC ofthesubstrate(Figure1.6).Bydelocalizingnegative
chargeatthiscenterPLPisabletomediatechemistryatthea-,b-,andc-centersof
appropriatelyfunctionalizedaminoacids(seeBox1.3).
Pyridoxal-dependent enzymes have been classified into five fold-types and the
aspartateaminotransferase(AATase)familyofenzymesbelongtoFold-TypeI[26,27].
ThecytosolicandmitochondrialAATaseswerethefirstPLP-dependentenzymesfor
whichdetailedstructuralinformationwasobtained[28–30].Theseenzymesinter-
convertglutamateandoxaloacetatewitha-KGandaspartate(Figure1.7).Glutamate
isactivatedbybindingtothePLPandthedisplacedSchiffbaseLys258actsasan
acid–base catalyst to transfer a proton between Ca and C40 of the PLP [31]. An
aspartate residue (Asp222) interacts with the protonated nitrogen of the cofactor,
R
H Enz
Enz H3N CO2 R H NH2 R H3NEnz
H N H N CO2 H N CO2
O O O
N OPO32 N OPO32 N OPO32
H H H
Enz-PLP
Figure1.6 SchiffbaseformationandanionstabilizationbyPLP-dependentenzymes.
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10 1 AminoAcidBiosynthesis
CO CO
2 2
H N CO H N CO
H 2 2
O + H O
–
N OPO 2 N OPO 2
3 3
O H O H
O O
CO Enz Enz CO
2 2
H N CO O CO
3 H 2 2
Glu Enz α-KG
H N H NH
2
O O
PLP form PMP form
N OPO 2 N OPO 2
3 3
O H O H
O O
Enz Enz
R R
R R
H N CO O CO
3 H 2 2
H N CO H N CO
α-Amino acid H 2 2 α-Keto acid
O + H O
–
N OPO 2 N OPO 2
3 3
O H O H
O O
Enz Enz
Figure1.7 Mechanismofaminotransferasecatalysis(forAATasesR¼CHCO(cid:3)).
2 2
stabilizingthepyridiniumformandfacilitatingdeprotonationofthesubstrate.Once
aprotonhasbeentransferred fromCatoC40,hydrolyticcleavageoftheketimine
linkage liberates a-KG and leaves the PMP form of the cofactor. Binding of
oxaloacetateandrunningthereactioninreverseleadstoregenerationoftheoriginal
enzymeandproductionofaspartate.Aminotransferaseenzymesprovideageneral
mechanismforinterconvertinga-aminoacidsanda-ketoacids,illustratingasecond
routebywhichnitrogenistransferredbetweenmetabolites.
1.3.2
CaseStudy:DualSubstrateSpecificityofFamiliesofAminotransferaseEnzymes
Aminotransferase enzymes pose an intriguing challenge for substrate specificity
since they bind two different substrates successively at the same site and must