Alteration of the Co-substrate Selectivity of Deacetoxycephalosporin C Synthase

Lee, Hwei-Jen; Lloyd, Matthew D.; Clifton, I.J.; Harlos, K.; Dubus, Alain; Baldwin, Jack E.; Frère, Jean Marie; Schofield, Christopher J.

doi:10.1074/jbc.m100085200

Cited by 39 publications

(34 citation statements)

References 49 publications

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“…Mutations to residues binding the iron(II), 2-oxoglutarate, or substrate have been demonstrated to cause uncoupled turnover of 2-oxoglutarate in other enzymes, e.g. DAOCS (10,11,13,14), and this has been ascribed to the mis-alignment of this high-energy iron species with respect to prime substrate. This phenomenon probably accounts for the behavior of the Trp-82 mutants reported here, suggesting that this residue affects the binding conformation of penicillin and deacetoxycephem substrates.…”

Section: Discussionmentioning

confidence: 99%

“…3), and 2-oxoglutarate co-substrate conversion (in the presence of both substrates). Both 2-oxoglutarate conversion and prime substrate oxidation (ring-expansion of penicillin G or hydroxylation of DAC) were measured as some oxygenase mutations can reduce or abolish prime substrate oxidation, whereas 2-oxoglutarate conversion can be retained ("uncoupled turnover") (10,11).…”

Section: Fig 1 Oxidative Reactions Carried Out By Daoc/dacs (C Acrmentioning

confidence: 99%

“…These studies have revealed the residues responsible for binding the iron(II) cofactor and the 2-oxoglutarate co-substrate (10,14), and these residues are strictly conserved in the DAOC/DACS and DACS sequences. The C terminus of DAOCS encloses the substrate in the active site and helps control activity (11).…”

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confidence: 99%

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Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase

Lloyd

Lipscomb²,

Hewitson³

et al. 2004

Journal of Biological Chemistry

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Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutaratedependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with sitedirected mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (⌬310 mutant) enhanced ring-expansion of penicillin G by ϳ2-fold. A double mutant, ⌬310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The ⌬310/N305L/ M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K m ‫؍‬ 2.00 ؎ 0.47 compared with 6.02 ؎ 0.97 mM for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.

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Section: Discussionmentioning

confidence: 99%

Section: Fig 1 Oxidative Reactions Carried Out By Daoc/dacs (C Acrmentioning

confidence: 99%

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Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase

Lloyd

Lipscomb²,

Hewitson³

et al. 2004

Journal of Biological Chemistry

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“…Henceforth, it will be easier to rationally modify the enzyme substrate preference to suit industrial needs. This has been illustrated by successful alteration of the co-substrate selectivity of scDAOCS [52]. The crystal structure of scDAOCS complexed with 2-oxoglutarate revealed that the 5-carboxylate of 2-oxoglutarate is stabilized by electrostatic interaction with the side chain of R258 [85].…”

Section: Alteration Of Deacetoxycephalosporin C Synthase Co-substratementioning

confidence: 95%

Directed evolution and rational approaches to improving Streptomyces clavuligerus deacetoxycephalosporin C synthase for cephalosporin production

Goo

Chua

Sim

2009

J Ind Microbiol Biotechnol

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It is approximately 60 years since the discovery of cephalosporin C in Cephalosporium acremonium. Streptomycetes have since been found to produce the structurally related cephamycin C. Studies on the biosynthetic pathways of these two compounds revealed a common pathway including a step governed by deacetoxycephalosporin C synthase which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C. Because of the therapeutic importance of cephalosporins, this enzyme has been extensively studied for its ability to produce these antibiotics. Although, on the basis of earlier studies, its substrate specificity was believed to be extremely narrow, relentless efforts in optimizing the in-vitro enzyme assay conditions showed that it is able to convert a wide range of penicillin substrates differing in their side chains. It is a member of 2-oxoglutarate-dependent dioxygenase protein family, which requires the iron(II) ion as a co-factor and 2-oxoglutarate and molecular oxygen as co-substrates. It has highly conserved HXDX( n ) H and RXS motifs to bind the co-factor and co-substrate, respectively. With advances in technology, the genes encoding this enzyme from various sources have been cloned and heterologously expressed for comparative analyses and mutagenesis studies. A high level of recombinant protein expression has also enabled crystallization of this enzyme for structure determination. This review will summarize some of the earlier biochemical characterization and describe the mechanistic action of this enzyme revealed by recent structural studies. This review will also discuss some of the approaches used to identify the amino acid residues involved in binding the penicillin substrate and to modify its substrate preference for possible industrial application.

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“…Replacement of Arg-36 with lysine had a similar effect as replacement with alanine, suggesting that, in addition to the positive charge at this position, the two-point interaction of the guanido group with Asp-34 is important. Chemical rescue of kinetic parameters with guanido derivatives has been reported for arginine replacement variants of several enzymes (2,8,20,21,27,29), including Cam, the prototype of the ␥-class carbonic anhydrase (39). These derivatives are thought to occupy the cavity vacated by the arginine side chain in a productive conformation which mimics the guanido group of arginine.…”

Section: Discussionmentioning

confidence: 99%

Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase

2002

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The roles of an aspartate and an arginine, which are completely conserved in the active sites of ␤-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the ␤-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the k cat /K m value depends only on the rate constants for the CO 2 hydration step, whereas k cat also depends on rate constants from the proton transfer step ( Carbonic anhydrase is a zinc-containing enzyme that catalyzes the reversible hydration of carbon dioxide:This enzyme, which is present in species from all three domains of life, plays a critical role in many diverse physiological processes such as respiration, photosynthesis, and CO 2 fixation. Based on amino acid sequence comparisons, carbonic anhydrases belong to four genetically distinct classes (␣, ␤, ␥, and ␦) of independent origins (38). The crystal structures for representatives of the ␣, ␤, and ␥ classes have now been determined (3, 7, 9-13, 16, 18, 19, 23, 26, 35, 36). Although the structure of the recently identified ␦-class prototype from the diatom Thalassiosira weissflogii has not yet been solved, extended Xray absorption fine-structure analyses suggest that the activesite zinc is coordinated by three histidines and one water molecule, as found in the ␣ and ␥ classes (6). The structures of the ␤-class carbonic anhydrases (7,18,26,36) indicate striking differences between this class and the others. For example, the active-site zinc in the ␤-class enzymes is coordinated by two cysteines, one histidine, and one water molecule.The kinetic properties of the human ␣-class isozymes have been comprehensively investigated and follow a common zinchydroxide mechanism for catalysis (24, 30). The catalytically active group in this mechanistic model is the zinc-bound water, which ionizes to a metal-bound hydroxyl which attacks CO 2 . Despite considerable structural differences in the active sites of these enzymes, the catalytic mechanisms of both ␥-and ␤-class enzymes resemble those of human ␣-class HCA II (1,14,15,28,31). The overall enzyme-catalyzed reaction occurs in two mechanistically distinct steps (where E ϭ enzyme and B ϭ buffer):The first step is the interconversion between carbon dioxide and bicarbonate (equations 2a and 2b) and involves the nucleophilic attack of the zinc-bound hydroxyl on the CO 2 molecule. In the ␣-class enzyme, the zinc-bound oxygen forms a hydrogen bond with 42,43). The role of this conserved gatekeeper residue is to prevent nonprotonated atoms from binding effectively. In addition, Thr-199 has been proposed to electrophilically activate the CO 2 molecule by forming a hydrogen bond with CO 2 through its backbone amide. The second step of the proposed mechanism is the regeneration of the zinc hydroxide at the active site of the enzyme (equations 2c and 2d), involving proton transfer events. The k cat /K m value depends only on the rate...

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Alteration of the Co-substrate Selectivity of Deacetoxycephalosporin C Synthase

Cited by 39 publications

References 49 publications

Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase

Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase

Directed evolution and rational approaches to improving Streptomyces clavuligerus deacetoxycephalosporin C synthase for cephalosporin production

Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase

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