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Jack E. Baldwin - One of the best experts on this subject based on the ideXlab platform.
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termiNally truNcated IsopeNicilliN N syNthase geNerates a dithioester product evideNce for a thioaldehyde iNtermediate duriNg catalysis aNd a New mode of reactioN for NoN heme iroN oxidases
Chemistry: A European Journal, 2017Co-Authors: Luke A Mcneill, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Nicolai I Burzlaff, Malkit Sami, Toby J N Brown, Timothy D W Claridge, Peter J RutledgeAbstract:IsopeNicilliN N syNthase (IPNS) catalyses the four-electroN oxidatioN of a tripeptide, l-δ-(α-amiNoadipoyl)-l-cysteiNyl-d-valiNe (ACV), to give IsopeNicilliN N (IPN), the first-formed β-lactam iN peNicilliN aNd cephalosporiN biosyNthesis. IPNS catalysis is depeNdeNt upoN aN iroN(II) cofactor aNd oxygeN as a co-substrate. IN the abseNce of substrate, the carboNyl oxygeN of the side-chaiN amide of the peNultimate residue, GlN330, co-ordiNates to the active-site metal iroN. Substrate biNdiNg ablates the iNteractioN betweeN GlN330 aNd the metal, triggeriNg rearraNgemeNt of seveN C-termiNal residues, which move to take up a coNformatioN that exteNds the fiNal α-helix aNd eNcloses ACV iN the active site. MutageNesis studies are reported, which probe the role of the C-termiNal aNd other aspects of the substrate biNdiNg pocket iN IPNS. The hydrophobic Nature of amiNo acid side-chaiNs arouNd the ACV biNdiNg pocket is importaNt iN catalysis. DeletioN of seveN C-termiNal residues exposes the active site aNd leads to formatioN of a New type of thiol oxidatioN product. The isolated product is showN by LC-MS aNd NMR aNalyses to be the eNe-thiol tautomer of a dithioester, made up from two molecules of ACV liNked betweeN the thiol sulfur of oNe tripeptide aNd the oxidised cysteiNyl β-carboN of the other. A mechaNism for its formatioN is proposed, supported by aN X-ray crystal structure, which shows the substrate ACV bouNd at the active site, its cysteiNyl β-carboN exposed to attack by a secoNd molecule of substrate, adjaceNt. FormatioN of this product coNstitutes a New mode of reactioN for IPNS aNd NoN-heme iroN oxidases iN geNeral.
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termiNally truNcated IsopeNicilliN N syNthase geNerates a dithioester product evideNce for a thioaldehyde iNtermediate duriNg catalysis aNd a New mode of reactioN for NoN heme iroN oxidases
Chemistry: A European Journal, 2017Co-Authors: Luke A Mcneill, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Nicolai I Burzlaff, Malkit Sami, Toby J N Brown, Timothy D W Claridge, Peter J RutledgeAbstract:IsopeNicilliN N syNthase (IPNS) catalyses the oxidatioN of a tripeptide, L-δ-(α-amiNoadipoyl)-L-cysteiNyl-D-valiNe (ACV), to IsopeNicilliN N (IPN), the first-formed β-lactam iN peNicilliN biosyNthesis. IPNS catalysis is depeNdeNt upoN aN iroN(II) cofactor aNd oxygeN as co-substrate. IN the abseNce of substrate, the carboNyl oxygeN of the side-chaiN amide of the peNultimate residue, GlN330, co-ordiNates to the active site metal. Substrate biNdiNg ablates this iNteractioN, triggeriNg rearraNgemeNt of seveN C-termiNal residues which move to take up a coNformatioN that exteNds the fiNal α-helix aNd eNcloses the active site. We report mutageNesis studies probiNg the role of the C-termiNal aNd other aspects of the substrate biNdiNg pocket iN IPNS. UNexpectedly, deletioN of seveN C-termiNal residues exposes the active site aNd leads to formatioN of a New type of thiol oxidatioN product. The isolated product is showN by LC-MS aNd NMR aNalyses to be the eNe-thiol tautomer of a dithioester, made up from two molecules of ACV liNked betweeN the thiol sulfur of oNe tripeptide aNd the oxidised cysteiNyl β-carboN of the other. A mechaNism for its formatioN is proposed, supported by X-ray crystal data which shows the substrate ACV bouNd at the active site, its cysteiNyl β-carboN exposed to attack by a secoNd molecule of substrate, adjaceNt. FormatioN of this product coNstitutes a New mode of reactioN for IPNS aNd NoN-heme iroN oxidases iN geNeral.
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the crystal structure of IsopeNicilliN N syNthase with a dipeptide substrate aNalogue
Archives of Biochemistry and Biophysics, 2013Co-Authors: Adam Daruzzaman, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Peter J RutledgeAbstract:Abstract IsopeNicilliN N syNthase (IPNS) coNverts its liNear tripeptide substrate δ- l -α-amiNoadipoyl- l -cysteiNyl- d -valiNe (ACV) to bicyclic IsopeNicilliN N (IPN), the key step iN peNicilliN biosyNthesis. SolutioN-phase iNcubatioN experimeNts have showN that IPNS will accept aNd oxidise a diverse array of substrate aNalogues, iNcludiNg tripeptides that iNcorporate l -homocysteiNe as their secoNd residue, aNd tripeptides with truNcated side-chaiNs at the third amiNo acid such as δ- l -α-amiNoadipoyl- l -cysteiNyl- d -α-amiNobutyrate (ACAb), δ- l -α-amiNoadipoyl- l -cysteiNyl- d -alaNiNe (ACA) aNd δ- l -α-amiNoadipoyl- l -cysteiNyl-glyciNe (ACG). However IPNS does Not react with dipeptide substrates. To probe this selectivity we have crystallised the eNzyme with the dipeptide δ- l -α-amiNoadipoyl- l -homocysteiNe (AhC) aNd solved a crystal structure for the IPNS:Fe(II):AhC complex to 1.40 A resolutioN. This structure reveals aN uNexpected mode of peptide biNdiNg at the IPNS active site, iN which the homocysteiNyl thiolate does Not biNd to iroN. INstead the primary mode of biNdiNg sees the homocysteiNyl carboxylate coordiNated to the metal, while its side-chaiN is orieNted iNto the regioN of the active site Normally occupied by the beNzyl group of proteiN residue Phe211.
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the crystal structure of IsopeNicilliN N syNthase with δ l α amiNoadipoyl l cysteiNyl d methioNiNe reveals thioether coordiNatioN to iroN
Archives of Biochemistry and Biophysics, 2011Co-Authors: Ian J Clifton, Robert M. Adlington, Jack E. Baldwin, Peter J RutledgeAbstract:Abstract IsopeNicilliN N syNthase (IPNS) catalyses cyclizatioN of δ-( l -α-amiNoadipoyl)- l -cysteiNyl- d -valiNe (ACV) to IsopeNicilliN N (IPN), the ceNtral step iN peNicilliN biosyNthesis. Previous studies have showN that IPNS turNs over a wide raNge of substrate aNalogues iN which the valiNe residue of its Natural substrate is replaced with other amiNo acids. IPNS accepts aNd oxidizes Numerous substrates that bear hydrocarboN sidechaiNs iN this positioN, however the eNzyme is less toleraNt of aNalogues preseNtiNg polar fuNctioNality iN place of the valiNyl isopropyl group. We report a New ACV aNalogue δ-( l -α-amiNoadipoyl)- l -cysteiNyl- d -methioNiNe (ACM), which iNcorporates a thioether iN place of the valiNyl sidechaiN. ACM has beeN syNthesized usiNg solutioN phase methods aNd crystallized with IPNS. A crystal structure has beeN elucidated for the IPNS:Fe(II):ACM complex at 1.40 A resolutioN. This structure reveals that ACM biNds iN the IPNS active site such that the sulfur atom of the methioNiNe thioether biNds to iroN iN the oxygeN biNdiNg site at a distaNce of 2.57 A. The sulfur of the cysteiNyl thiolate sits 2.36 A from the metal.
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crystallographic studies oN the biNdiNg of selectively deuterated lld aNd lll substrate epimers by IsopeNicilliN N syNthase
Biochemical and Biophysical Research Communications, 2010Co-Authors: Ian J Clifton, Robert M. Adlington, Jack E. Baldwin, Jeanette E Stok, Peter J RutledgeAbstract:Abstract IsopeNicilliN N syNthase (IPNS) is a NoN-heme iroN(II) oxidase which catalyses the biosyNthesis of IsopeNicilliN N (IPN) from the tripeptide δ- l -α-amiNoadipoyl- l -cysteiNyl- d -valiNe ( lld -ACV). HereiN we report crystallographic studies to iNvestigate the biNdiNg of a truNcated lll -substrate iN the active site of IPNS. Two epimeric tripeptides have beeN prepared by solutioN phase peptide syNthesis aNd crystallised with the eNzyme. δ- l -α-AmiNoadipoyl- l -cysteiNyl- d -2-amiNo-3,3-dideuteriobutyrate ( lld -AC d 2 Ab) has the same coNfiguratioN as the Natural substrate lld -ACV at each of its three stereoceNtres; its epimer δ- l -α-amiNoadipoyl- l -cysteiNyl- l -2-amiNo-3,3-dideuteriobutyrate ( lll -AC d 2 Ab) has the opposite coNfiguratioN at its third amiNo acid. lll -ACV has previously beeN showN to iNhibit IPNS turNover of its substrate lld -ACV; the all-protiated tripeptide δ- l -α-amiNoadipoyl- l -cysteiNyl- d -2-amiNobutyrate ( lld -ACAb) is a substrate for IPNS, beiNg turNed over to a mixture of peNam aNd cepham products. ComparisoNs betweeN the crystal structures of the IPNS:Fe(II): lld -AC d 2 Ab aNd IPNS:Fe(II): lll -AC d 2 Ab complexes offer a possible ratioNale for the previously observed iNhibitory effects of lll -ACV oN IPNS activity.
Peter J Rutledge - One of the best experts on this subject based on the ideXlab platform.
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IsopeNicilliN N syNthase crystallographic studies
ChemBioChem, 2021Co-Authors: Nicole C Chapman, Peter J RutledgeAbstract:IsopeNicilliN N syNthase (IPNS) is a NoN-heme iroN oxidase (NHIO) that catalyses the cyclisatioN of tripeptide δ-(l-α-amiNoadipoyl)-l-cysteiNyl-d-valiNe (ACV) to bicyclic IsopeNicilliN N (IPN). Over the last 25 years, crystallography has shed coNsiderable light oN the mechaNism of IPNS catalysis. The first crystal structure, for apo-IPNS with MN bouNd iN place of Fe at the active site, reported iN 1995, was also the first structure for a member of the wider NHIO family. This was followed by the aNaerobic eNzyme-substrate complex IPNS-Fe-ACV (1997), this complex plus Nitric oxide as a surrogate for co-substrate dioxygeN (1997), aNd aN eNzyme product complex (1999). SiNce theN, crystallography has beeN used to probe maNy aspects of the IPNS reactioN mechaNism, by crystallisiNg the proteiN with a diversity of substrate aNalogues aNd triggeriNg the oxidative reactioN by usiNg elevated oxygeN pressures to force the gaseous co-substrate throughout proteiN crystals aNd maximise syNchroNicity of turNover iN crystallo. IN this way, X-ray structures have beeN elucidated for a raNge of complexes closely related to aNd/or directly derived from key iNtermediates iN the catalytic cycle, thereby aNsweriNg Numerous mechaNistic questioNs that had ariseN from solutioN-phase experimeNts, aNd posiNg maNy New oNes. The results of these crystallographic studies have, iN turN, iNformed computatioNal experimeNts that have brought further iNsight. These combiNed crystallographic aNd computatioNal iNvestigatioNs augmeNt aNd exteNd the results of earlier spectroscopic aNalyses aNd solutioN phase studies of IPNS turNover, to eNrich our uNderstaNdiNg of this importaNt proteiN aNd the wider NHIO eNzyme family.
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termiNally truNcated IsopeNicilliN N syNthase geNerates a dithioester product evideNce for a thioaldehyde iNtermediate duriNg catalysis aNd a New mode of reactioN for NoN heme iroN oxidases
Chemistry: A European Journal, 2017Co-Authors: Luke A Mcneill, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Nicolai I Burzlaff, Malkit Sami, Toby J N Brown, Timothy D W Claridge, Peter J RutledgeAbstract:IsopeNicilliN N syNthase (IPNS) catalyses the four-electroN oxidatioN of a tripeptide, l-δ-(α-amiNoadipoyl)-l-cysteiNyl-d-valiNe (ACV), to give IsopeNicilliN N (IPN), the first-formed β-lactam iN peNicilliN aNd cephalosporiN biosyNthesis. IPNS catalysis is depeNdeNt upoN aN iroN(II) cofactor aNd oxygeN as a co-substrate. IN the abseNce of substrate, the carboNyl oxygeN of the side-chaiN amide of the peNultimate residue, GlN330, co-ordiNates to the active-site metal iroN. Substrate biNdiNg ablates the iNteractioN betweeN GlN330 aNd the metal, triggeriNg rearraNgemeNt of seveN C-termiNal residues, which move to take up a coNformatioN that exteNds the fiNal α-helix aNd eNcloses ACV iN the active site. MutageNesis studies are reported, which probe the role of the C-termiNal aNd other aspects of the substrate biNdiNg pocket iN IPNS. The hydrophobic Nature of amiNo acid side-chaiNs arouNd the ACV biNdiNg pocket is importaNt iN catalysis. DeletioN of seveN C-termiNal residues exposes the active site aNd leads to formatioN of a New type of thiol oxidatioN product. The isolated product is showN by LC-MS aNd NMR aNalyses to be the eNe-thiol tautomer of a dithioester, made up from two molecules of ACV liNked betweeN the thiol sulfur of oNe tripeptide aNd the oxidised cysteiNyl β-carboN of the other. A mechaNism for its formatioN is proposed, supported by aN X-ray crystal structure, which shows the substrate ACV bouNd at the active site, its cysteiNyl β-carboN exposed to attack by a secoNd molecule of substrate, adjaceNt. FormatioN of this product coNstitutes a New mode of reactioN for IPNS aNd NoN-heme iroN oxidases iN geNeral.
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termiNally truNcated IsopeNicilliN N syNthase geNerates a dithioester product evideNce for a thioaldehyde iNtermediate duriNg catalysis aNd a New mode of reactioN for NoN heme iroN oxidases
Chemistry: A European Journal, 2017Co-Authors: Luke A Mcneill, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Nicolai I Burzlaff, Malkit Sami, Toby J N Brown, Timothy D W Claridge, Peter J RutledgeAbstract:IsopeNicilliN N syNthase (IPNS) catalyses the oxidatioN of a tripeptide, L-δ-(α-amiNoadipoyl)-L-cysteiNyl-D-valiNe (ACV), to IsopeNicilliN N (IPN), the first-formed β-lactam iN peNicilliN biosyNthesis. IPNS catalysis is depeNdeNt upoN aN iroN(II) cofactor aNd oxygeN as co-substrate. IN the abseNce of substrate, the carboNyl oxygeN of the side-chaiN amide of the peNultimate residue, GlN330, co-ordiNates to the active site metal. Substrate biNdiNg ablates this iNteractioN, triggeriNg rearraNgemeNt of seveN C-termiNal residues which move to take up a coNformatioN that exteNds the fiNal α-helix aNd eNcloses the active site. We report mutageNesis studies probiNg the role of the C-termiNal aNd other aspects of the substrate biNdiNg pocket iN IPNS. UNexpectedly, deletioN of seveN C-termiNal residues exposes the active site aNd leads to formatioN of a New type of thiol oxidatioN product. The isolated product is showN by LC-MS aNd NMR aNalyses to be the eNe-thiol tautomer of a dithioester, made up from two molecules of ACV liNked betweeN the thiol sulfur of oNe tripeptide aNd the oxidised cysteiNyl β-carboN of the other. A mechaNism for its formatioN is proposed, supported by X-ray crystal data which shows the substrate ACV bouNd at the active site, its cysteiNyl β-carboN exposed to attack by a secoNd molecule of substrate, adjaceNt. FormatioN of this product coNstitutes a New mode of reactioN for IPNS aNd NoN-heme iroN oxidases iN geNeral.
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the crystal structure of IsopeNicilliN N syNthase with a dipeptide substrate aNalogue
Archives of Biochemistry and Biophysics, 2013Co-Authors: Adam Daruzzaman, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Peter J RutledgeAbstract:Abstract IsopeNicilliN N syNthase (IPNS) coNverts its liNear tripeptide substrate δ- l -α-amiNoadipoyl- l -cysteiNyl- d -valiNe (ACV) to bicyclic IsopeNicilliN N (IPN), the key step iN peNicilliN biosyNthesis. SolutioN-phase iNcubatioN experimeNts have showN that IPNS will accept aNd oxidise a diverse array of substrate aNalogues, iNcludiNg tripeptides that iNcorporate l -homocysteiNe as their secoNd residue, aNd tripeptides with truNcated side-chaiNs at the third amiNo acid such as δ- l -α-amiNoadipoyl- l -cysteiNyl- d -α-amiNobutyrate (ACAb), δ- l -α-amiNoadipoyl- l -cysteiNyl- d -alaNiNe (ACA) aNd δ- l -α-amiNoadipoyl- l -cysteiNyl-glyciNe (ACG). However IPNS does Not react with dipeptide substrates. To probe this selectivity we have crystallised the eNzyme with the dipeptide δ- l -α-amiNoadipoyl- l -homocysteiNe (AhC) aNd solved a crystal structure for the IPNS:Fe(II):AhC complex to 1.40 A resolutioN. This structure reveals aN uNexpected mode of peptide biNdiNg at the IPNS active site, iN which the homocysteiNyl thiolate does Not biNd to iroN. INstead the primary mode of biNdiNg sees the homocysteiNyl carboxylate coordiNated to the metal, while its side-chaiN is orieNted iNto the regioN of the active site Normally occupied by the beNzyl group of proteiN residue Phe211.
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the crystal structure of IsopeNicilliN N syNthase with δ l α amiNoadipoyl l cysteiNyl d methioNiNe reveals thioether coordiNatioN to iroN
Archives of Biochemistry and Biophysics, 2011Co-Authors: Ian J Clifton, Robert M. Adlington, Jack E. Baldwin, Peter J RutledgeAbstract:Abstract IsopeNicilliN N syNthase (IPNS) catalyses cyclizatioN of δ-( l -α-amiNoadipoyl)- l -cysteiNyl- d -valiNe (ACV) to IsopeNicilliN N (IPN), the ceNtral step iN peNicilliN biosyNthesis. Previous studies have showN that IPNS turNs over a wide raNge of substrate aNalogues iN which the valiNe residue of its Natural substrate is replaced with other amiNo acids. IPNS accepts aNd oxidizes Numerous substrates that bear hydrocarboN sidechaiNs iN this positioN, however the eNzyme is less toleraNt of aNalogues preseNtiNg polar fuNctioNality iN place of the valiNyl isopropyl group. We report a New ACV aNalogue δ-( l -α-amiNoadipoyl)- l -cysteiNyl- d -methioNiNe (ACM), which iNcorporates a thioether iN place of the valiNyl sidechaiN. ACM has beeN syNthesized usiNg solutioN phase methods aNd crystallized with IPNS. A crystal structure has beeN elucidated for the IPNS:Fe(II):ACM complex at 1.40 A resolutioN. This structure reveals that ACM biNds iN the IPNS active site such that the sulfur atom of the methioNiNe thioether biNds to iroN iN the oxygeN biNdiNg site at a distaNce of 2.57 A. The sulfur of the cysteiNyl thiolate sits 2.36 A from the metal.
Ian J Clifton - One of the best experts on this subject based on the ideXlab platform.
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termiNally truNcated IsopeNicilliN N syNthase geNerates a dithioester product evideNce for a thioaldehyde iNtermediate duriNg catalysis aNd a New mode of reactioN for NoN heme iroN oxidases
Chemistry: A European Journal, 2017Co-Authors: Luke A Mcneill, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Nicolai I Burzlaff, Malkit Sami, Toby J N Brown, Timothy D W Claridge, Peter J RutledgeAbstract:IsopeNicilliN N syNthase (IPNS) catalyses the oxidatioN of a tripeptide, L-δ-(α-amiNoadipoyl)-L-cysteiNyl-D-valiNe (ACV), to IsopeNicilliN N (IPN), the first-formed β-lactam iN peNicilliN biosyNthesis. IPNS catalysis is depeNdeNt upoN aN iroN(II) cofactor aNd oxygeN as co-substrate. IN the abseNce of substrate, the carboNyl oxygeN of the side-chaiN amide of the peNultimate residue, GlN330, co-ordiNates to the active site metal. Substrate biNdiNg ablates this iNteractioN, triggeriNg rearraNgemeNt of seveN C-termiNal residues which move to take up a coNformatioN that exteNds the fiNal α-helix aNd eNcloses the active site. We report mutageNesis studies probiNg the role of the C-termiNal aNd other aspects of the substrate biNdiNg pocket iN IPNS. UNexpectedly, deletioN of seveN C-termiNal residues exposes the active site aNd leads to formatioN of a New type of thiol oxidatioN product. The isolated product is showN by LC-MS aNd NMR aNalyses to be the eNe-thiol tautomer of a dithioester, made up from two molecules of ACV liNked betweeN the thiol sulfur of oNe tripeptide aNd the oxidised cysteiNyl β-carboN of the other. A mechaNism for its formatioN is proposed, supported by X-ray crystal data which shows the substrate ACV bouNd at the active site, its cysteiNyl β-carboN exposed to attack by a secoNd molecule of substrate, adjaceNt. FormatioN of this product coNstitutes a New mode of reactioN for IPNS aNd NoN-heme iroN oxidases iN geNeral.
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termiNally truNcated IsopeNicilliN N syNthase geNerates a dithioester product evideNce for a thioaldehyde iNtermediate duriNg catalysis aNd a New mode of reactioN for NoN heme iroN oxidases
Chemistry: A European Journal, 2017Co-Authors: Luke A Mcneill, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Nicolai I Burzlaff, Malkit Sami, Toby J N Brown, Timothy D W Claridge, Peter J RutledgeAbstract:IsopeNicilliN N syNthase (IPNS) catalyses the four-electroN oxidatioN of a tripeptide, l-δ-(α-amiNoadipoyl)-l-cysteiNyl-d-valiNe (ACV), to give IsopeNicilliN N (IPN), the first-formed β-lactam iN peNicilliN aNd cephalosporiN biosyNthesis. IPNS catalysis is depeNdeNt upoN aN iroN(II) cofactor aNd oxygeN as a co-substrate. IN the abseNce of substrate, the carboNyl oxygeN of the side-chaiN amide of the peNultimate residue, GlN330, co-ordiNates to the active-site metal iroN. Substrate biNdiNg ablates the iNteractioN betweeN GlN330 aNd the metal, triggeriNg rearraNgemeNt of seveN C-termiNal residues, which move to take up a coNformatioN that exteNds the fiNal α-helix aNd eNcloses ACV iN the active site. MutageNesis studies are reported, which probe the role of the C-termiNal aNd other aspects of the substrate biNdiNg pocket iN IPNS. The hydrophobic Nature of amiNo acid side-chaiNs arouNd the ACV biNdiNg pocket is importaNt iN catalysis. DeletioN of seveN C-termiNal residues exposes the active site aNd leads to formatioN of a New type of thiol oxidatioN product. The isolated product is showN by LC-MS aNd NMR aNalyses to be the eNe-thiol tautomer of a dithioester, made up from two molecules of ACV liNked betweeN the thiol sulfur of oNe tripeptide aNd the oxidised cysteiNyl β-carboN of the other. A mechaNism for its formatioN is proposed, supported by aN X-ray crystal structure, which shows the substrate ACV bouNd at the active site, its cysteiNyl β-carboN exposed to attack by a secoNd molecule of substrate, adjaceNt. FormatioN of this product coNstitutes a New mode of reactioN for IPNS aNd NoN-heme iroN oxidases iN geNeral.
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the crystal structure of IsopeNicilliN N syNthase with a dipeptide substrate aNalogue
Archives of Biochemistry and Biophysics, 2013Co-Authors: Adam Daruzzaman, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Peter J RutledgeAbstract:Abstract IsopeNicilliN N syNthase (IPNS) coNverts its liNear tripeptide substrate δ- l -α-amiNoadipoyl- l -cysteiNyl- d -valiNe (ACV) to bicyclic IsopeNicilliN N (IPN), the key step iN peNicilliN biosyNthesis. SolutioN-phase iNcubatioN experimeNts have showN that IPNS will accept aNd oxidise a diverse array of substrate aNalogues, iNcludiNg tripeptides that iNcorporate l -homocysteiNe as their secoNd residue, aNd tripeptides with truNcated side-chaiNs at the third amiNo acid such as δ- l -α-amiNoadipoyl- l -cysteiNyl- d -α-amiNobutyrate (ACAb), δ- l -α-amiNoadipoyl- l -cysteiNyl- d -alaNiNe (ACA) aNd δ- l -α-amiNoadipoyl- l -cysteiNyl-glyciNe (ACG). However IPNS does Not react with dipeptide substrates. To probe this selectivity we have crystallised the eNzyme with the dipeptide δ- l -α-amiNoadipoyl- l -homocysteiNe (AhC) aNd solved a crystal structure for the IPNS:Fe(II):AhC complex to 1.40 A resolutioN. This structure reveals aN uNexpected mode of peptide biNdiNg at the IPNS active site, iN which the homocysteiNyl thiolate does Not biNd to iroN. INstead the primary mode of biNdiNg sees the homocysteiNyl carboxylate coordiNated to the metal, while its side-chaiN is orieNted iNto the regioN of the active site Normally occupied by the beNzyl group of proteiN residue Phe211.
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the crystal structure of IsopeNicilliN N syNthase with δ l α amiNoadipoyl l cysteiNyl d methioNiNe reveals thioether coordiNatioN to iroN
Archives of Biochemistry and Biophysics, 2011Co-Authors: Ian J Clifton, Robert M. Adlington, Jack E. Baldwin, Peter J RutledgeAbstract:Abstract IsopeNicilliN N syNthase (IPNS) catalyses cyclizatioN of δ-( l -α-amiNoadipoyl)- l -cysteiNyl- d -valiNe (ACV) to IsopeNicilliN N (IPN), the ceNtral step iN peNicilliN biosyNthesis. Previous studies have showN that IPNS turNs over a wide raNge of substrate aNalogues iN which the valiNe residue of its Natural substrate is replaced with other amiNo acids. IPNS accepts aNd oxidizes Numerous substrates that bear hydrocarboN sidechaiNs iN this positioN, however the eNzyme is less toleraNt of aNalogues preseNtiNg polar fuNctioNality iN place of the valiNyl isopropyl group. We report a New ACV aNalogue δ-( l -α-amiNoadipoyl)- l -cysteiNyl- d -methioNiNe (ACM), which iNcorporates a thioether iN place of the valiNyl sidechaiN. ACM has beeN syNthesized usiNg solutioN phase methods aNd crystallized with IPNS. A crystal structure has beeN elucidated for the IPNS:Fe(II):ACM complex at 1.40 A resolutioN. This structure reveals that ACM biNds iN the IPNS active site such that the sulfur atom of the methioNiNe thioether biNds to iroN iN the oxygeN biNdiNg site at a distaNce of 2.57 A. The sulfur of the cysteiNyl thiolate sits 2.36 A from the metal.
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crystallographic studies oN the biNdiNg of selectively deuterated lld aNd lll substrate epimers by IsopeNicilliN N syNthase
Biochemical and Biophysical Research Communications, 2010Co-Authors: Ian J Clifton, Robert M. Adlington, Jack E. Baldwin, Jeanette E Stok, Peter J RutledgeAbstract:Abstract IsopeNicilliN N syNthase (IPNS) is a NoN-heme iroN(II) oxidase which catalyses the biosyNthesis of IsopeNicilliN N (IPN) from the tripeptide δ- l -α-amiNoadipoyl- l -cysteiNyl- d -valiNe ( lld -ACV). HereiN we report crystallographic studies to iNvestigate the biNdiNg of a truNcated lll -substrate iN the active site of IPNS. Two epimeric tripeptides have beeN prepared by solutioN phase peptide syNthesis aNd crystallised with the eNzyme. δ- l -α-AmiNoadipoyl- l -cysteiNyl- d -2-amiNo-3,3-dideuteriobutyrate ( lld -AC d 2 Ab) has the same coNfiguratioN as the Natural substrate lld -ACV at each of its three stereoceNtres; its epimer δ- l -α-amiNoadipoyl- l -cysteiNyl- l -2-amiNo-3,3-dideuteriobutyrate ( lll -AC d 2 Ab) has the opposite coNfiguratioN at its third amiNo acid. lll -ACV has previously beeN showN to iNhibit IPNS turNover of its substrate lld -ACV; the all-protiated tripeptide δ- l -α-amiNoadipoyl- l -cysteiNyl- d -2-amiNobutyrate ( lld -ACAb) is a substrate for IPNS, beiNg turNed over to a mixture of peNam aNd cepham products. ComparisoNs betweeN the crystal structures of the IPNS:Fe(II): lld -AC d 2 Ab aNd IPNS:Fe(II): lll -AC d 2 Ab complexes offer a possible ratioNale for the previously observed iNhibitory effects of lll -ACV oN IPNS activity.
Robert M. Adlington - One of the best experts on this subject based on the ideXlab platform.
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termiNally truNcated IsopeNicilliN N syNthase geNerates a dithioester product evideNce for a thioaldehyde iNtermediate duriNg catalysis aNd a New mode of reactioN for NoN heme iroN oxidases
Chemistry: A European Journal, 2017Co-Authors: Luke A Mcneill, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Nicolai I Burzlaff, Malkit Sami, Toby J N Brown, Timothy D W Claridge, Peter J RutledgeAbstract:IsopeNicilliN N syNthase (IPNS) catalyses the four-electroN oxidatioN of a tripeptide, l-δ-(α-amiNoadipoyl)-l-cysteiNyl-d-valiNe (ACV), to give IsopeNicilliN N (IPN), the first-formed β-lactam iN peNicilliN aNd cephalosporiN biosyNthesis. IPNS catalysis is depeNdeNt upoN aN iroN(II) cofactor aNd oxygeN as a co-substrate. IN the abseNce of substrate, the carboNyl oxygeN of the side-chaiN amide of the peNultimate residue, GlN330, co-ordiNates to the active-site metal iroN. Substrate biNdiNg ablates the iNteractioN betweeN GlN330 aNd the metal, triggeriNg rearraNgemeNt of seveN C-termiNal residues, which move to take up a coNformatioN that exteNds the fiNal α-helix aNd eNcloses ACV iN the active site. MutageNesis studies are reported, which probe the role of the C-termiNal aNd other aspects of the substrate biNdiNg pocket iN IPNS. The hydrophobic Nature of amiNo acid side-chaiNs arouNd the ACV biNdiNg pocket is importaNt iN catalysis. DeletioN of seveN C-termiNal residues exposes the active site aNd leads to formatioN of a New type of thiol oxidatioN product. The isolated product is showN by LC-MS aNd NMR aNalyses to be the eNe-thiol tautomer of a dithioester, made up from two molecules of ACV liNked betweeN the thiol sulfur of oNe tripeptide aNd the oxidised cysteiNyl β-carboN of the other. A mechaNism for its formatioN is proposed, supported by aN X-ray crystal structure, which shows the substrate ACV bouNd at the active site, its cysteiNyl β-carboN exposed to attack by a secoNd molecule of substrate, adjaceNt. FormatioN of this product coNstitutes a New mode of reactioN for IPNS aNd NoN-heme iroN oxidases iN geNeral.
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termiNally truNcated IsopeNicilliN N syNthase geNerates a dithioester product evideNce for a thioaldehyde iNtermediate duriNg catalysis aNd a New mode of reactioN for NoN heme iroN oxidases
Chemistry: A European Journal, 2017Co-Authors: Luke A Mcneill, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Nicolai I Burzlaff, Malkit Sami, Toby J N Brown, Timothy D W Claridge, Peter J RutledgeAbstract:IsopeNicilliN N syNthase (IPNS) catalyses the oxidatioN of a tripeptide, L-δ-(α-amiNoadipoyl)-L-cysteiNyl-D-valiNe (ACV), to IsopeNicilliN N (IPN), the first-formed β-lactam iN peNicilliN biosyNthesis. IPNS catalysis is depeNdeNt upoN aN iroN(II) cofactor aNd oxygeN as co-substrate. IN the abseNce of substrate, the carboNyl oxygeN of the side-chaiN amide of the peNultimate residue, GlN330, co-ordiNates to the active site metal. Substrate biNdiNg ablates this iNteractioN, triggeriNg rearraNgemeNt of seveN C-termiNal residues which move to take up a coNformatioN that exteNds the fiNal α-helix aNd eNcloses the active site. We report mutageNesis studies probiNg the role of the C-termiNal aNd other aspects of the substrate biNdiNg pocket iN IPNS. UNexpectedly, deletioN of seveN C-termiNal residues exposes the active site aNd leads to formatioN of a New type of thiol oxidatioN product. The isolated product is showN by LC-MS aNd NMR aNalyses to be the eNe-thiol tautomer of a dithioester, made up from two molecules of ACV liNked betweeN the thiol sulfur of oNe tripeptide aNd the oxidised cysteiNyl β-carboN of the other. A mechaNism for its formatioN is proposed, supported by X-ray crystal data which shows the substrate ACV bouNd at the active site, its cysteiNyl β-carboN exposed to attack by a secoNd molecule of substrate, adjaceNt. FormatioN of this product coNstitutes a New mode of reactioN for IPNS aNd NoN-heme iroN oxidases iN geNeral.
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the crystal structure of IsopeNicilliN N syNthase with a dipeptide substrate aNalogue
Archives of Biochemistry and Biophysics, 2013Co-Authors: Adam Daruzzaman, Robert M. Adlington, Jack E. Baldwin, Ian J Clifton, Peter J RutledgeAbstract:Abstract IsopeNicilliN N syNthase (IPNS) coNverts its liNear tripeptide substrate δ- l -α-amiNoadipoyl- l -cysteiNyl- d -valiNe (ACV) to bicyclic IsopeNicilliN N (IPN), the key step iN peNicilliN biosyNthesis. SolutioN-phase iNcubatioN experimeNts have showN that IPNS will accept aNd oxidise a diverse array of substrate aNalogues, iNcludiNg tripeptides that iNcorporate l -homocysteiNe as their secoNd residue, aNd tripeptides with truNcated side-chaiNs at the third amiNo acid such as δ- l -α-amiNoadipoyl- l -cysteiNyl- d -α-amiNobutyrate (ACAb), δ- l -α-amiNoadipoyl- l -cysteiNyl- d -alaNiNe (ACA) aNd δ- l -α-amiNoadipoyl- l -cysteiNyl-glyciNe (ACG). However IPNS does Not react with dipeptide substrates. To probe this selectivity we have crystallised the eNzyme with the dipeptide δ- l -α-amiNoadipoyl- l -homocysteiNe (AhC) aNd solved a crystal structure for the IPNS:Fe(II):AhC complex to 1.40 A resolutioN. This structure reveals aN uNexpected mode of peptide biNdiNg at the IPNS active site, iN which the homocysteiNyl thiolate does Not biNd to iroN. INstead the primary mode of biNdiNg sees the homocysteiNyl carboxylate coordiNated to the metal, while its side-chaiN is orieNted iNto the regioN of the active site Normally occupied by the beNzyl group of proteiN residue Phe211.
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the crystal structure of IsopeNicilliN N syNthase with δ l α amiNoadipoyl l cysteiNyl d methioNiNe reveals thioether coordiNatioN to iroN
Archives of Biochemistry and Biophysics, 2011Co-Authors: Ian J Clifton, Robert M. Adlington, Jack E. Baldwin, Peter J RutledgeAbstract:Abstract IsopeNicilliN N syNthase (IPNS) catalyses cyclizatioN of δ-( l -α-amiNoadipoyl)- l -cysteiNyl- d -valiNe (ACV) to IsopeNicilliN N (IPN), the ceNtral step iN peNicilliN biosyNthesis. Previous studies have showN that IPNS turNs over a wide raNge of substrate aNalogues iN which the valiNe residue of its Natural substrate is replaced with other amiNo acids. IPNS accepts aNd oxidizes Numerous substrates that bear hydrocarboN sidechaiNs iN this positioN, however the eNzyme is less toleraNt of aNalogues preseNtiNg polar fuNctioNality iN place of the valiNyl isopropyl group. We report a New ACV aNalogue δ-( l -α-amiNoadipoyl)- l -cysteiNyl- d -methioNiNe (ACM), which iNcorporates a thioether iN place of the valiNyl sidechaiN. ACM has beeN syNthesized usiNg solutioN phase methods aNd crystallized with IPNS. A crystal structure has beeN elucidated for the IPNS:Fe(II):ACM complex at 1.40 A resolutioN. This structure reveals that ACM biNds iN the IPNS active site such that the sulfur atom of the methioNiNe thioether biNds to iroN iN the oxygeN biNdiNg site at a distaNce of 2.57 A. The sulfur of the cysteiNyl thiolate sits 2.36 A from the metal.
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crystallographic studies oN the biNdiNg of selectively deuterated lld aNd lll substrate epimers by IsopeNicilliN N syNthase
Biochemical and Biophysical Research Communications, 2010Co-Authors: Ian J Clifton, Robert M. Adlington, Jack E. Baldwin, Jeanette E Stok, Peter J RutledgeAbstract:Abstract IsopeNicilliN N syNthase (IPNS) is a NoN-heme iroN(II) oxidase which catalyses the biosyNthesis of IsopeNicilliN N (IPN) from the tripeptide δ- l -α-amiNoadipoyl- l -cysteiNyl- d -valiNe ( lld -ACV). HereiN we report crystallographic studies to iNvestigate the biNdiNg of a truNcated lll -substrate iN the active site of IPNS. Two epimeric tripeptides have beeN prepared by solutioN phase peptide syNthesis aNd crystallised with the eNzyme. δ- l -α-AmiNoadipoyl- l -cysteiNyl- d -2-amiNo-3,3-dideuteriobutyrate ( lld -AC d 2 Ab) has the same coNfiguratioN as the Natural substrate lld -ACV at each of its three stereoceNtres; its epimer δ- l -α-amiNoadipoyl- l -cysteiNyl- l -2-amiNo-3,3-dideuteriobutyrate ( lll -AC d 2 Ab) has the opposite coNfiguratioN at its third amiNo acid. lll -ACV has previously beeN showN to iNhibit IPNS turNover of its substrate lld -ACV; the all-protiated tripeptide δ- l -α-amiNoadipoyl- l -cysteiNyl- d -2-amiNobutyrate ( lld -ACAb) is a substrate for IPNS, beiNg turNed over to a mixture of peNam aNd cepham products. ComparisoNs betweeN the crystal structures of the IPNS:Fe(II): lld -AC d 2 Ab aNd IPNS:Fe(II): lll -AC d 2 Ab complexes offer a possible ratioNale for the previously observed iNhibitory effects of lll -ACV oN IPNS activity.
Christopher J Schofield - One of the best experts on this subject based on the ideXlab platform.
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roles of 2 oxoglutarate oxygeNases aNd IsopeNicilliN N syNthase iN β lactam biosyNthesis
Natural Product Reports, 2018Co-Authors: Patrick Rabe, Jos J A G Kamps, Christopher J Schofield, Christopher T LohansAbstract:CoveriNg: up to 2017 2-Oxoglutarate (2OG) depeNdeNt oxygeNases aNd the homologous oxidase IsopeNicilliN N syNthase (IPNS) play crucial roles iN the biosyNthesis of β-lactam riNg coNtaiNiNg Natural products. IPNS catalyses formatioN of the bicyclic peNicilliN Nucleus from a tripeptide. 2OG oxygeNases catalyse reactioNs that diversify the chemistry of β-lactams formed by both IPNS aNd NoN-oxidative eNzymes. ReactioNs catalysed by the 2OG oxygeNases of β-lactam biosyNthesis Not oNly iNvolve their typical hydroxylatioN reactioNs, but also desaturatioN, epimerisatioN, rearraNgemeNt, aNd riNg-formiNg reactioNs. Some of the eNzymes iNvolved iN β-lactam biosyNthesis exhibit remarkable substrate aNd product selectivities. We review the roles of 2OG oxygeNases aNd IPNS iN β-lactam biosyNthesis, highlightiNg opportuNities for applicatioN of kNowledge of their roles, structures, aNd mechaNisms.
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crystallizatioN aNd prelimiNary x ray diffractioN studies oN recombiNaNt IsopeNicilliN N syNthase from aspergillus NidulaNs
Protein Science, 2008Co-Authors: Peter L Roach, Jack E. Baldwin, Ian J Clifton, Christopher J Schofield, Janos HajduAbstract:RecombiNaNt Aspergillus NidulaNs IsopeNicilliN N syNthase was purified from aN Escherichia coli expressioN system. The apoeNzyme iN the preseNce of saturatiNg coNceNtratioNs of MNCl2 could be crystallized by either macro- or microseediNg, usiNg the haNgiNg drop vapor diffusioN techNique with polyethyleNe glycol 8000 as precipitaNt. The crystals (0.5-1.0 mm overall dimeNsioNs) diffract X-rays to at least 2.0 A resolutioN at syNchrotroNs aNd beloNg to space group P212121 with uNit cell dimeNsioNs of a = 59.2 A, b = 127.0 A, aNd c = 139.6 A. The asymmetric uNit coNtaiNs oNe dimer, aNd the solveNt coNteNt of the crystals is 60%. The crystals are radiatioN seNsitive.
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structural studies oN the reactioN of IsopeNicilliN N syNthase with the truNcated substrate aNalogues delta l alpha amiNoadipoyl l cysteiNyl glyciNe aNd delta l alpha amiNoadipoyl l cysteiNyl d alaNiNe
Biochemistry, 2005Co-Authors: Alexandra J Long, Jack E. Baldwin, Ian J Clifton, Peter J Rutledge, Peter L Roach, Christopher J SchofieldAbstract:IsopeNicilliN N syNthase (IPNS), a NoN-heme iroN(II)-depeNdeNt oxidase, catalyzes coNversioN of the tripeptide delta-(l-alpha-amiNoadipoyl)-l-cysteiNyl-d-valiNe (ACV) to bicyclic IsopeNicilliN N (IPN), coNcomitaNt with the reductioN of dioxygeN to two molecules of water. INcubatioN of the "truNcated"substrate aNalogues delta-(l-alpha-amiNoadipoyl)-l-cysteiNyl-glyciNe (ACG) aNd delta-(l-alpha-amiNoadipoyl)-l-cysteiNyl-d-alaNiNe (ACA) with IPNS has previously beeN showN to afford acyclic products, iN which the substrate cysteiNyl residue has uNdergoNe a two-electroN oxidatioN. We report X-ray crystal structures for the aNaerobic IPNS/Fe(II)/ACG aNd IPNS/Fe(II)/ACA complexes, both iN the abseNce aNd preseNce of the dioxygeN aNalogue Nitric oxide. The overall proteiN structures are very similar to those of the correspoNdiNg IPNS/Fe(II)/ACV complexes; however, sigNificaNt differeNces are appareNt iN the viciNity of the active site iroN. The structure of the IPNS/Fe(II)/ACG complex reveals that the C-termiNal carboxylate of this substrate is orieNted toward the active site iroN atom, appareNtly hydrogeN-boNded to aN additioNal water ligaNd at the metal; this is a differeNt biNdiNg mode to that observed iN the IPNS/Fe(II)/ACV complex. ACA biNds to the metal iN a maNNer that is iNtermediate betweeN those observed for ACV aNd ACG. The additioN of NO to these complexes iNitiates coNformatioNal chaNges such that both the IPNS/Fe(II)/ACG/NO aNd IPNS/Fe(II)/ACA/NO structures closely resemble the IPNS/Fe(II)/ACV/NO complex. These results further demoNstrate the feasibility of metal-ceNtered rearraNgemeNts iN catalysis by NoN-heme iroN eNzymes aNd provide iNsight iNto the delicate balaNce betweeN hydrophilic-hydrophobic iNteractioNs aNd steric effects iN the IPNS active site.
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structural studies oN the reactioN of IsopeNicilliN N syNthase with the substrate aNalogue delta l alpha amiNoadipoyl l cysteiNyl d alpha amiNobutyrate
Biochemical Journal, 2003Co-Authors: Alexandra J Long, Jack E. Baldwin, Ian J Clifton, Christopher J Schofield, Peter L Roach, Peter J RutledgeAbstract:IsopeNicilliN N syNthase (IPNS) is a NoN-haem iroN(II) oxidase which catalyses the biosyNthesis of IsopeNicilliN N from the tripeptide delta-(L-alpha-amiNoadipoyl)-L-cysteiNyl-D-valiNe (ACV). HereiN we report crystallographic studies to iNvestigate the reactioN of IPNS with the truNcated substrate aNalogue delta-(L-alpha-amiNoadipoyl)-L-cysteiNyl-D-alpha-amiNobutyrate (ACAb). It has beeN reported previously that this aNalogue gives rise to three beta-lactam products wheN iNcubated with IPNS: two methyl peNams aNd a cepham. Crystal structures of the IPNS-Fe(II)-ACAb aNd IPNS-Fe(II)-ACAb-NO complexes have Now beeN solved aNd are reported hereiN. These structures aNd modelliNg studies based oN them shed light oN the dimiNished product selectivity showN by IPNS iN its reactioN with ACAb aNd further ratioNalize the preseNce of certaiN key residues at the IPNS active site.
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studies of IsopeNicilliN N syNthase eNzymatic properties usiNg a coNtiNuous spectrophotometric assay
FEBS Letters, 2000Co-Authors: Alain Dubus, Jack E. Baldwin, Christopher J Schofield, Malkit Sami, Toby J N Brown, Jeanmarie FrereAbstract:IsopeNicilliN N syNthase (IPNS) from Aspergillus NidulaNs is a No-heme iroN(II)-depeNdeNt oxygeNase which catalyses, iN a siNgle reactioN, the bicyclisatioN of delta-(L-alpha-amiNoadipoyl)-L-cysteiNyl-D-valiNe iNto IsopeNicilliN N, the precursor of all other peNicilliNs, cephalosporiNs aNd cephamyciNs. The IPNS reactioN caN be followed directly aNd coNtiNuously by a New assay which moNitors the absorbaNce iNcrease at 235 Nm characteristic of peNicilliN Nucleus formatioN. UsiNg this assay, the effects of iNflueNtial factors affectiNg the iN vitro IPNS eNzymatic reactioN were iNvestigated. EveN uNder optimal coNditioNs, eNzyme iNactivatioN occurred duriNg catalysis. IroN(II) depletioN aNd product iNhibitioN were Not the cause of this pheNomeNoN, the additioN of aNtioxidaNts or reduciNg ageNts failed to slow dowN iNactivatioN or reactivate the eNzyme. Therefore, this pheNomeNoN appears to be irreversible aNd is attributed to oxidative damage caused to the eNzyme by reactive oxygeN species geNerated iN solutioN duriNg catalysis. Nevertheless, the steady-state kiNetic parameters for the IPNS reactioN were determiNed.
