University of Groningen
The hole story. Structure and function of the 70 kDa soluble lytic transglycosylase
from escherichia coli
Thunnissen, Andy
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Publication date:
1995
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Thunnissen, A-M. W. H. (1995). The hole story. Structure and function of the 70 kDa soluble lytic
transglycosylase from escherichia coli Groningen: s.n.
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133
Summary
Structural integrity of the bacterialcell wall is of vital importancefor the prokaryotic cell.
Without a rigid, intact cell wall, bacteriaare no longer able to resist the high osmotic
pressureinsidetheir cells:bulgesform at the cell surfaceand,eventually,the bacteriawill
burst. The strengthand rigidity of the cell wall is definedby a unique molecularmesh
built up of linearpolysaccharide
This is a heteropolymer
known as mureinor peptidoglycan.
strands, cross-linked by short peptide chains, forming a network-like structure that
surroundsthe cell as one giant macromolecule.The polysaccharide(glycan) strandsare
(GlcNAc) and N-acetylcomposedof two alternatingsugarresidues,N-acetylglucosamine
bonds.The peptidesare
muramicacid (MurNAc), which areconnectedby B-1,4-glycosidic
linked to the glycanchainsvia the lactyl groupof the MurNAc residuesandcontaina small
number of both L and D-amino acid residues.A typical peptide in the cell wall
peptidoglycanof Escherichiacoli consistof .L-alanine-D-isoglutamyl-n-diaminopimelyl-Dalanine(L-Ala-D-Glu-m-Dap-D - Ala).
The integrityand shapeof the peptidoglycanduringgrowth andcell divisionarethoughtto
and peptidoglycanbe preservedby a balancedaction of pepidoglycan-synthesising
degradingenzymes.It is a challengeto understandthe natureof this interplayof enzymes.
Such knowledge would be directly applicablefor the developmentof antibiotica.The
in the synthesisof the
antibacterialactivity of penicillin,for instance,is dueto a disturbance
peptidecross-linkages
in the peptidoglycan.Althoughpenicillin is still the most useddrug
for treatmentof bacterialinfections,its potencyhasbeen severelydiminisheddue to the
developmentof antibiotic resistancein bacteria.This so called "crisis of antibiotic
resistance"hasrevivedthe researchinterestin the peptidoglycanmetabolicpathwayswith
the objectiveto identify new targetsfor the designof novel antibiotics,distinct from the
from Escherichiacoli.
penicillins.Suchpotentialnew targetsarethe lytic transglycosylases
enzymesinvolved in bacterialcell wall
These are endogenouspeptidoglycan-degrading
growth and turnover.The lytic transglycosylases
catalysethe cleavageof the B-1,4glycosidic bondsin the peptidoglycan,an activity that is unexploitedby most naturaland
syntheticantibiotics.The activity is similarto that of the lysozymes,a well-studiedclassof
enzymes.However,while the Iysozymescleavethe
anti-bacterialpeptidoglycan-degrading
peptidoglycanby hydrolysisof the glycosidicbonds,the lytic transglycosylases
accomplish
this by catalysinga glycosyl transferasereactionwhereby new, 1,6-anhydrobonds are
lbrmed in the MurNAc residues.Furthermore.while lvsozvmeis an endo-muramidase
with
134
generalaccessto the glycosidic bonds in the peptidoglycannetwork, SLT70 is an exomuramidasethat can cleavethe glycosidicbondsonly startingfrom the looseendsof the
glycanstands.
This thesisdescribesthe determinationof the crystalstructureof the 70-kDa solublelytic
transglycosylasefrom E. coli. The three-dimensionalstructureprovides the framework
necessaryfor studyingthe catalyticpropertiesof the enzyme.As a long-termobjectivethe
structuremay be usedfor the designof specificinhibitors and novel antibiotics.
Chapter 1 providesan overviewof the currentstateof knowledgeconcerningthe bacterial
peptidoglycanand the lytic transglycosylases.
In spite of extensivebiochemicaland
biophysicalstudies,little is known about the precisemechanismsthat play a role in the
assembly,growth and turnoverof the bacterialcell wall. The physiologicalrole and the
arestill elusive.The mechanismof action
reactionmechanismof the lytic transglycosylase
of lysozyme, on the other hand, is rather well defined, and forms a referencefor the
analysis of SLT70. In the reaction mechanismof lysozyme two amino acids play an
importantrole: a glutamic acid and an aspartateresidue.The glutamic acid startsthe cleavage
reactionby donatingaproton to the centraloxygenatomin the scissile,glycosidicbond.As
an intermediatestep,a very labile,positivelychargedglycosyloxocarboniumion is formed,
that is stabilisedby the negativechargeof the aspartate.In the final stepof the reaction this
oxocarboniumion is attackedby a water molecule,releasingthe cleavedsugarfrom the
activesiteof the enzyme.
In chapters 2 and 3 the three-dimensional
structureof SLT70 is introduced.The structure
was determinedby X-ray crystallographyat a resolutionof 2.1 j\. The SLT70 protein
moleculeturnedout to havean extraordinaryshape.Insteadof globular,like most proteins,
SLT70 is doughnutshaped,with a largeholein the middle.Eiectronmicroscopycodfirmed
the unusualshapeof the SLT70 protein.The SLT70 doughnutis built up of threedomains.
The first 360 amino acid residuesform a ratherextendedU-shapeddomain.By a loop of
-20 amino acidsthis U-domainis connectedto the linker or L-domain (70 amino acids).
This seconddomainclosesthe mouth of the U-domain,therebyforming a centralhole of
more than 25 A in diameter.The first two domainsof SLT70 containonly cr-helice
s,27 in
total, which are folded into an unusualright-handedsuperhelix.The superhelixmay be
describedas two neighbouringlayersof cr-helices;within eachlayer helicesrun parallel,
while betweenlayersanti-parallelhelix pairsareformed.This arrangementis quite unique
and differs from that observedin the "standard"4-cr-helixbundle,in which all helix pairs
are anti-parallel.So far, only one protein is known with a domain of similar fold: the
lipovellinefrom the eggsof lamprey.
Summary
135
On top of the superhelicalring lies the third, C-terminaldomain,suchthat the centralhole
to solvent.This C-domainconsistsof nine a-helicesand a small
remainslargely accessible
antiparallel p-sheet.Its fold resemblesthat of lysozyme,althoughthere is no obvious
By site-directedmutagensisand an inhibitor binding
relationshipin amino acid sequence.
study it could be shown that the C-domaincontainsthe activesite of SLT70 with Glu478 as
the equivalentof the "catalytic"glutamicacid in lysozyme.However,a "catalytic"aspartate
is not able
is absentin the activesite of SLT70.This impliesthat the lytic transglycosylase
to providethe samestabilisationof a possibleoxocarboniumintermediate
aslysozymedoes.
This is not unreasonable,though,since the inffamolecularglycosyl transferasereaction
catalysedby SLT70 probably requiresa shorterlifetime of the oxocarboniumion compared
to the hydrolysisreactioncatalysedby lysozyme.The functionof the U and L-domainsin
SLT70 is unknown.Probablythey are involved in the binding of peptidoglycan.It is also
conceivable that the ring structure of these domains forms a steric obstruction that
determinesthe exolyticactivityof SLT70.
Chapter 4 describesa detailedstructuralcomparisonof the C-domainof SLT70 with the
three-dimensionalstructuresof three different lysozymes:chicken-typehen egg-white
lysozyme, goose-type swan egg-white lysozyme and phage-typelysozyme from
a distinctclassin the lysozymefamily
bacteriopahe
T4. Eachof theselysozymesrepresents
on the basisof amino acid sequenceand catalyticactivity.Our investigationsrevealedthat
the C-terminal domain of SLT70 defines a novel bacterial class of lysozymesthat is
characterisedby the absenceof a "catalytic"aspartatein the activesite and a small numberof
differences in secondarystructureelements.The best agreementin three-dimensional
structureis found with the goose-typelysozyme,which interestingly,alsomissesa catalytic
aspartate
in the activesite.
In chapters 5 and 6 the resultsof threecrystal binding studiesof SLT70 are presented.
The experimentswere carried out by soakingcrystalsof SLT70 in solutionscontaining
compounds.The compoundsusedincludeda glycopeptide
different peptidoglycan-related
with a GlcNAc residueand a substitutedprolinederivative,a trisaccharide
of only GlcNAc
residuesand a glycopeptideconsistingof a GlcNAc and a 1,6-anhydroMurNAcresidue
linked to an l-Ala-D-Glu-zr-Dap peptide.The first compoundis also known as bulgecin
and the cornplexof SLT70 with this specificinhibitor was mentionedbeforein chapter2.
The secondcompoundis a fragmentof chitin,a polymerfrom the cell wall of yeast.The last
compoundis a naturalreactionproductof SLT70. All threecompoundsbind to the Cdomainof SLT70, in a groovethat alsocontainsthe activesite of the enzyme.Analysisof
the structLlres
of the three complexesconfirms that SLT70, like lysozyme,utilisesan
extended,peptidoglycanbindingsitethat can accomodate
five to six consecutivesaccharide
136
residues.Furthermore,the resultsshow that the enzymealso has a binding site for the
peptidemoietiesin the peptidoglycan.
The role of Glu478as catalyticacid is confirmedby
the SLT70 complexes.The SlT70-bulgecinis especiallyinterestingbecausewith its
positively chargedproline derivativethe inhibitor could mimick the conformationof the
oxocarboniumreactionintermediate
of SLT70.
The results describedin this thesisform a basis for further researchon the action and
function of SLT70. Severalquestionsremainto be answered.What function have the first
two domainsin the SLT70 doughnut?Which differencesin the catalyticcentreof SLT70,in
addition to the absenceof a catalytic aspartate,determinethat SLT70 acts as a lytic
transglycosylase,
insteadof a lysozyme.What mechanismenablesthe SLT70 doughnutto
pass the peptide cross-bridgeswhen it moves along the glycan strands.Structure
determinationand analysisof SLT70 mutantscomplexedwith larger fragmentsof
peptidoglycanareessentialfor answeringthesequestions.Futureresearchon the structure
and function of SLT70 shouldestablishwhetherthe enzymeis suitablefor the desisn of
novel antibiotics.