Cell-free biosynthesis of penicillins. Conversion of peptides into new .beta.-lactam antibiotics

Bahadur, Gulam; Baldwin, Jack E.; Usher, John J.; Abraham, E. P.; Jayatilake, Gamini S.; White, Robert L.

doi:10.1021/ja00415a041

Cited by 41 publications

(25 citation statements)

References 2 publications

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“…A) The reaction of ACI (4) with IPNS gives penam 6, while its epimer ACaI 5 gives the epimeric penam 7. [10,11] (ACI also gives rise to small amounts of two epimeric 2-methyl-cepham products, in ca. 10 % abundance relative to penam 6.…”

Section: Structure Of the Ipns:fementioning

confidence: 99%

“…However IPNS is much less tolerant of polar side chains and heteroatoms in this position. [6] Thus, whereas d-l-a-aminoadipoyl-l-cysteinyl-d-isoleucine (ACI, 4) and AC-d-allo-isoleucine (ACaI, 5) are converted by IPNS to the penam products 6 and 7 respectively (Scheme 2), [10][11][12] the isosteric sulfur-containing peptides AC-d-thiaisoleucine (ACtI, 8) and AC-d-thia-allo-isoleucine (ACtaI, 9) are not turned over. [6] To investigate the failure of IPNS to turn over such substrates, we have synthesised the tripeptide ACtaI 9, crystallised it with IPNS, and solved the structure of the resulting complex.…”

Section: Introductionmentioning

confidence: 99%

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Isopenicillin N Synthase Binds δ‐(L‐α‐Aminoadipoyl)‐L‐Cysteinyl‐D‐Thia‐allo‐Isoleucine through both Sulfur Atoms

Clifton¹,

Ge²,

Adlington³

et al. 2011

ChemBioChem

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Isopenicillin N synthase (IPNS) catalyses the synthesis of isopenicillin N (IPN), the biosynthetic precursor to penicillin and cephalosporin antibiotics. IPNS is a non-heme iron(II) oxidase that mediates the oxidative cyclisation of the tripeptide δ-L-α-aminoadipoyl-L-cysteinyl-D-valine (ACV) to IPN with a concomitant reduction of molecular oxygen to water. Solution-phase incubation experiments have shown that, although IPNS can turn over analogues with a diverse range of hydrocarbon side chains in the third (valinyl) position of its substrate, the enzyme is much less tolerant of polar residues in this position. Thus, although IPNS converts δ-L-α-aminoadipoyl-L-cysteinyl-D-isoleucine (ACI) and AC-D-allo-isoleucine (ACaI) to penam products, the isosteric sulfur-containing peptides AC-D-thiaisoleucine (ACtI) and AC-D-thia-allo-isoleucine (ACtaI) are not turned over. To determine why these peptides are not substrates, we crystallized ACtaI with IPNS. We report the synthesis of ACtaI and the crystal structure of the IPNS:Fe(II) :ACtaI complex to 1.79 Å resolution. This structure reveals direct ligation of the thioether side chain to iron: the sulfide sulfur sits 2.66 Å from the metal, squarely in the oxygen binding site. This result articulates a structural basis for the failure of IPNS to turn over these substrates.

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Section: Structure Of the Ipns:fementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Isopenicillin N Synthase Binds δ‐(L‐α‐Aminoadipoyl)‐L‐Cysteinyl‐D‐Thia‐allo‐Isoleucine through both Sulfur Atoms

Clifton¹,

Ge²,

Adlington³

et al. 2011

ChemBioChem

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“…The results observed with AmCOV are indirectly supported by incubation studies with the substrates d-(La-aminoadipoyl)-L-cysteinyl-D-isoleucine (ACI) [33] and d-(L-a-aminoadipoyl)-L-cysteinyl-D-aminobutyrate (ACAb). [34][35][36] On incubation with IPNS, ACI gave a mixture of penam and cepham products. Formation of the penam can be rationalised by considering abstraction of hydrogen from the isoleucine b-carbon atom, generating a tertiary radical which then reacts with the sulfur.…”

Section: A Mechanism For Product Formationmentioning

confidence: 99%

Unexpected Oxidation of a Depsipeptide Substrate Analogue in Crystalline Isopenicillin N Synthase

et al. 2006

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Isopenicillin N synthase (IPNS) is a non-heme iron(ii)-dependent oxidase that is central to penicillin biosynthesis. Herein, we report mechanistic studies of the IPNS reaction in the crystalline state, using the substrate analogue delta-(L-alpha-aminoadipoyl)-(3R)-methyl-L-cysteine D-alpha-hydroxyisovaleryl ester (AmCOV) to probe the early stages of the catalytic cycle. The X-ray crystal structure of the anaerobic IPNS:Fe(II):AmCOV complex was solved to 1.40 A resolution, and it reveals several subtle differences in the active site relative to the complex of the enzyme with its natural substrate. The crystalline IPNS:Fe(II):AmCOV complex was then exposed to oxygen gas at high pressure; this brought about reaction to give what appears to be a hydroxymethyl/ene-thiol product. A mechanism for this reaction is proposed. These results offer further insight into the delicate interplay of steric and electronic effects in the IPNS active site and the mechanistic intricacies of this remarkable enzyme.

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“…Investigations into the substrate specificity of the cyclizing enzyme of C . acremonium have already indicated this enzyme to have a narrow substrate specificity (6,9,14).…”

Section: Introductionmentioning

confidence: 99%

Penicillin formation by cell-free extracts of Streptomycesclavuligerus. Behaviour of aminoadipyl-modified analogs of the natural peptide precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV)

Jensen¹,

Westlake²,

Bowers³

et al. 1984

Can. J. Chem.

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. Can. J. Chem. 62, 27 12 (1984). Six analogs of ACV, the biosynthetic precursor of the penicillin nucleus, have been synthesized, in which the 6-(L-a-aminoadipyl) moiety has been replaced by : P-(L-aspartyl), y-(L-glutamyl), 6-(D-a-aminoadipyl), adipyl, glycyl-6-(L-a-aminoadipyl), and N-acetyl-6-(L-a-aminoadipyl). The penicillins having the adipyl, glycyl-6-(L-a-aminoadipyl), and N-acetyl-6-(L-a-aminoadipyl) side chains have also been synthesized. Several improvements have been made in earlier routes to ACV and related peptides, including a simplified preparation of a dipeptide precursor, and a new synthesis of a-aminoadipic acid from lysine. A new reagent, 2-acetoximino-2-phenylacetonitrile, has been synthesized, which allows ready N-acetylation of amino hydroxyacids in aqueous acetone at room temperature. The peptide analogs have been examined as substrates of the enzyme isopenicillin N synthetase, which converts ACV to isopenicillin N in the presence of oxygen, ferrous ions, and ascorbate. The enzyme has been isolated from the prokaryotic organism Streptomyces clavuligerus, and has been employed either as a crude salt precipitate or in semi-purified form, freed from other enzymes of the penicillin-cephalosporin pathway. With the crude enzyme preparation, three of the analogs are active substrates, viz., adipyl, glycyl-6-(L-a-aminoadipyl), and N-acetyl-6-(L-a-aminoadipyl), but the latter two are converted, only via ACV, to isopenicillin N, the normal cyclization product. That the crude enzyme preparation contains a protease that deacylates N-substituted ACV analogs to ACV is confinned, inter alia, by the behaviour of the purified enzyme; of the various analogs, only the adipyl compound is an active substrate, but the conversion of this analog to carboxybutylpenicillin proceeds with only 1 -2%.efficiency, compared to the natural substrate. On a synthCtisC six analogues de I'ACV, le precurseur biosynthttique du noyau de la pknicilline, dans lesquel on a remplacC I'unitC 6-(L-a-aminoadipyl) par les unitts suivantes: P-(L-aspartyl), y-(L-glutamyl), 6-(D-a-aminoadipyl), adipyl, glycyl-6-(L-a-aminoadipyl) et N-acCtyl-6-(L-a-aminoadipyl). On a Cgalement synthCtisC des ptnicillines ayant des groupes adipyl, glycyl-6-(L-a-aminoadipyl) et N-acCtyl-6-(L-a-aminoadipyl) comme chaines lattrales. On a apportt plusieurs ameliorations aux voies d'accts a 1'ACV et aux peptides apparent& que nous avions dtcrites anttrieurement; ces amiliorations comprennent entre autre une prkparation simplifiCe d'un dipeptide prkcurseur ainsi qu'une nouvelle synthtse de I'acide a-aminoadipique a partir de la lysine. On a synthCtisC un nouveau reactif, I'acCtoxymino-2 phenyl-2 acitonitrile, qui permet d'effectuer facilement une N-acCtylation des hydroxy-acides amints en opirant dans l'acttone aqueuse et a la temperature ambiante. On a CtudiC le comportement des analogues peptidiques comme substrats de l'enzyme isopCnicilline N-synthktase qui transforme I'ACV en iso@nicilline N en presence d'oxygkne, d'ions ferreux et d'ions ascorbate. On a isol...

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Cell-free biosynthesis of penicillins. Conversion of peptides into new .beta.-lactam antibiotics

Cited by 41 publications

References 2 publications

Isopenicillin N Synthase Binds δ‐(L‐α‐Aminoadipoyl)‐L‐Cysteinyl‐D‐Thia‐allo‐Isoleucine through both Sulfur Atoms

Isopenicillin N Synthase Binds δ‐(L‐α‐Aminoadipoyl)‐L‐Cysteinyl‐D‐Thia‐allo‐Isoleucine through both Sulfur Atoms

Unexpected Oxidation of a Depsipeptide Substrate Analogue in Crystalline Isopenicillin N Synthase

Penicillin formation by cell-free extracts of Streptomycesclavuligerus. Behaviour of aminoadipyl-modified analogs of the natural peptide precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV)

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