Theputativediamine N-acetyltransferaseD2023.4hasbeen clonedfromthemodelnematode Caenorhabditiselegans.The 483bpopenreadingframeofthecDNAencodesadeducedpoly- peptideof18.6kDa.Accordingly,therecombinantlyexpressed His6-taggedproteinformsanenzymicallyactivehomodimerwith amolecularmassofapprox.44000Da.Theproteinbelongsto theGNAT(GCN5-related N-acetyltransferase)superfamily,and itsaminoacidsequenceexhibitsconsiderablesimilaritytomam- malianspermidine/spermine-N1 -acetyltransferases.However, neitherthepolyaminesspermidineandsperminenorthediamines putrescineandcadaverinewereefficientlyacetylatedbythepro- tein.Thesmallerdiaminesdiaminopropaneandethylenediamine, aswellas L-lysine,representbettersubstrates,but,surprisingly, theenzymemostefficientlycatalysesthe N-acetylationofamino acidsanalogouswith L-lysine.Asdeterminedbythe kcat /Km values,the C.elegansN-acetyltransferaseprefersthialysine [S-(2-aminoethyl)-L-cysteine],followedby O-(2-aminoethyl)-L-serineand S-(2-aminoethyl)-D,L-homocysteine.Reversed-phase HPLCandmassspectrometricanalysesrevealedthat N-acetyl- ationof L-lysineand L-thialysineoccursexclusivelyattheamino moietyofthesidechain.Remarkably,heterologousexpression of C.elegansN-acetyltransferaseD2023.4in Escherichiacoli, whichdoesnotpossessahomologousgene,resultsinapro- nouncedresistanceagainsttheanti-metabolitethialysine.Further- more,C.elegansN-acetyltransferaseD2023.4exhibitsthehighest homologywithanumberofGNATsfoundinnumerousgenomes frombacteriatomammalsthathavenotbeenbiochemically characterizedsofar,suggestinganovelgroupofGNATenzymes closelyrelatedtospermidine/spermine-N1 -acetyltransferase,but withadistinctsubstratespecificity.Takentogether,weproposeto nametheenzyme‘thialysine Nε -acetyltransferase’.
A novel member of the GCN5-related N-acetyltransferase superfamily from Caenorhabditis elegans preferentially catalyses the N-acetylation of thialysine (S-(2-aminoethyl)-L-cysteine)
PINNEN, Francesco Enrico;
2004-01-01
Abstract
Theputativediamine N-acetyltransferaseD2023.4hasbeen clonedfromthemodelnematode Caenorhabditiselegans.The 483bpopenreadingframeofthecDNAencodesadeducedpoly- peptideof18.6kDa.Accordingly,therecombinantlyexpressed His6-taggedproteinformsanenzymicallyactivehomodimerwith amolecularmassofapprox.44000Da.Theproteinbelongsto theGNAT(GCN5-related N-acetyltransferase)superfamily,and itsaminoacidsequenceexhibitsconsiderablesimilaritytomam- malianspermidine/spermine-N1 -acetyltransferases.However, neitherthepolyaminesspermidineandsperminenorthediamines putrescineandcadaverinewereefficientlyacetylatedbythepro- tein.Thesmallerdiaminesdiaminopropaneandethylenediamine, aswellas L-lysine,representbettersubstrates,but,surprisingly, theenzymemostefficientlycatalysesthe N-acetylationofamino acidsanalogouswith L-lysine.Asdeterminedbythe kcat /Km values,the C.elegansN-acetyltransferaseprefersthialysine [S-(2-aminoethyl)-L-cysteine],followedby O-(2-aminoethyl)-L-serineand S-(2-aminoethyl)-D,L-homocysteine.Reversed-phase HPLCandmassspectrometricanalysesrevealedthat N-acetyl- ationof L-lysineand L-thialysineoccursexclusivelyattheamino moietyofthesidechain.Remarkably,heterologousexpression of C.elegansN-acetyltransferaseD2023.4in Escherichiacoli, whichdoesnotpossessahomologousgene,resultsinapro- nouncedresistanceagainsttheanti-metabolitethialysine.Further- more,C.elegansN-acetyltransferaseD2023.4exhibitsthehighest homologywithanumberofGNATsfoundinnumerousgenomes frombacteriatomammalsthathavenotbeenbiochemically characterizedsofar,suggestinganovelgroupofGNATenzymes closelyrelatedtospermidine/spermine-N1 -acetyltransferase,but withadistinctsubstratespecificity.Takentogether,weproposeto nametheenzyme‘thialysine Nε -acetyltransferase’.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
