Studies on the mechanism of deacetoxy–deacetylcephalosporin C synthase using cyclopropyl substituted cephalosporin probes

Baldwin, Jack E.; Adlington, Robert M.; Crouch, Nicholas P.; Keeping, John W.; Leppard, Simon W.; Pitlik, J.; Schofield, Christopher J.; Sobey, Wendy J.; Wood, Mark E.

doi:10.1039/c39910000768

Cited by 17 publications

(23 citation statements)

References 18 publications

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“…See text for details. The hydroxyation step is shown as a radical rebound mechanism in accord with studies on prolyl‐4‐hydroylase and other 2‐OG oxygenases [55,56]. The binding and release of substrates and products is probably accompanied by significant conformational changes between ‘open’ and ‘closed’ forms of the active site [21].…”

Section: Resultsmentioning

confidence: 56%

Structure of proline 3‐hydroxylase

Clifton¹,

Hsueh²,

Baldwin³

et al. 2001

European Journal of Biochemistry

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101

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Iron (II)/2-oxoglutarate (2-OG)-dependent oxygenases catalyse oxidative reactions in a range of metabolic processes including the hydroxylation of proline and lysine residues during the post-translational modification of collagen. 2-OG oxygenases commonly require ascorbate for full activity. In the vitamin C deficient disease, scurvy, reduced activity of 2-OG oxygenases results in impaired formation of collagen. Here we report the crystal structure of bacterial proline 3-hydroxylase from Streptomyces sp., an enzyme which hydroxylates proline at position 3, the first of a 2-OG oxygenase catalysing oxidation of a free a-amino acid. Structures were obtained for the enzyme in the absence of iron (to 2.3Å resolution, R ¼ 20.2%, R free ¼ 25.3%) and that complexed to iron (II) (to 2.4Å resolution, R ¼ 19.8%, R free ¼ 22.6%). The structure contains conserved motifs present in other 2-OG oxygenases including a 'jelly roll' b strand core and residues binding iron and 2-oxoglutarate, consistent with divergent evolution within the extended family. The structure differs significantly from many other 2-OG oxygenases in possessing a discrete C-terminal helical domain. Analysis of the structure suggests a model for proline binding and a mechanism for uncoupling of proline and 2-OG turnover.

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Section: Resultsmentioning

confidence: 56%

Structure of proline 3‐hydroxylase

Clifton¹,

Hsueh²,

Baldwin³

et al. 2001

European Journal of Biochemistry

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101

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“…For some 2OG dependent oxygenases, including taurine dioxygenase and deacetoxycephalosporin C synthase, kinetic isotope studies are consistent with a mechanism in which a ferryl intermediate abstracts a hydrogen atom from the prime substrate. 25,26 This can be followed by substrate hydroxylation, desaturation or rearrangement. Desaturation can be envisaged to occur either by initial oxygenation followed by dehydration or via a more direct process not involving oxygenation (Fig.…”

Section: Discussionmentioning

confidence: 99%

Structural and mechanistic studies on anthocyanidin synthase catalysed oxidation of flavanone substrates: the effect of C-2 stereochemistry on product selectivity and mechanism

Welford¹,

Clifton²,

Turnbull³

et al. 2005

Org. Biomol. Chem.

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During the biosynthesis of the tricyclic flavonoid natural products in plants, oxidative modifications to the central C-ring are catalysed by Fe(ii) and 2-oxoglutarate dependent oxygenases. The reactions catalysed by three of these enzymes; flavone synthase I, flavonol synthase and anthocyanidin synthase (ANS), are formally desaturations. In comparison, flavanone 3beta-hydroxylase catalyses hydroxylation at the C-3 pro-R position of 2S-naringenin. Incubation of ANS with the unnatural substrate (+/-)-naringenin results in predominantly C-3 hydroxylation to give cis-dihydrokaempferol as the major product; trans-dihydrokaempferol and the desaturation product, apigenin are also observed. Labelling studies have demonstrated that some of the formal desaturation reactions catalysed by ANS proceed via initial C-3 hydroxylation followed by dehydration at the active site. We describe analyses of the reaction of ANS with 2S- and 2R-naringenin substrates, including the anaerobic crystal structure of an ANS-Fe-2-oxoglutarate-naringenin complex. Together the results reveal that for the 'natural' C-2 stereochemistry of 2S-naringenin, C-3 hydroxylation predominates (>9 : 1) over desaturation, probably due to the inaccessibility of the C-2 hydrogen to the iron centre. For the 2R-naringenin substrate, desaturation is significantly increased relative to C-3 hydroxylation (ca. 1 : 1); this is probably a result of both the C-3 pro-S and C-2 hydrogen atoms being accessible to the reactive oxidising intermediate in this substrate. In contrast to the hydroxylation-elimination desaturation mechanism for some ANS substrates, the results imply that the ANS catalysed desaturation of 2R-naringenin to form apigenin proceeds with a syn-arrangement of eliminated hydrogen atoms and not via an oxygenated (gem-diol) flavonoid intermediate. Thus, by utilising flavonoid substrates with different C-2 stereochemistries, the balance between C-3 hydroxylation or C-2, C-3 desaturation mechanisms can be altered.

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“…102 This result has been supported by studies with cyclopropyl derivatives of cephems with purified enzyme and biomimetic systems. 103 Deuterium labelling of the penicillin nucleus at C-2 resulted in the production of the 3-hydroxy-3methylcepham, 104,105 suggesting the desaturation occurred as a later step in the reaction. Studies on purified DAOCS indicated that the pro-S methyl group of 3 was also incorporated into the cephem ring of 4.…”

Section: Penicillin Cephalosporin and Cephamycin Biosynthesismentioning

confidence: 99%

“…These subtle mechanistic nuances may be a consequence of the greater versatility in oxidation chemistry exhibited by sulfur in the IPNS substrate compared to that of carbon or oxygen atoms which are more typically oxidised. In the case of the 2-oxoglutarate-dependent oxygenases, several studies have indicated that a radical mechanism is utilised by some enzymes, including DAOCS 103 and BBH. 211 It has been recently proposed that CAS utilises a radical (or carbenium) mechanism in the oxidative cyclisation of 11 to 12 (Scheme 2).…”

Section: Alkaloid Biosynthesismentioning

confidence: 99%