Penicillins and cephalosporins are among the most widely used therapeutic agents. These antibiotics are produced from fermentation-derived materials as their chemical synthesis is not commercially viable. Unconventional steps in their biosynthesis are catalysed by Fe(II)-dependent oxidases/oxygenases; isopenicillin N synthase (IPNS) creates in one step the bicyclic nucleus of penicillins, and deacetoxycephalosporin C synthase (DAOCS) catalyses the expansion of the penicillin nucleus into the nucleus of cephalosporins. Both enzymes use dioxygen-derived ferryl intermediates in catalysis but, in contrast to IPNS, the ferryl form of DAOCS is produced by the oxidative splitting of a co-substrate, 2-oxoglutarate (alpha-ketoglutarate). This route of controlled ferryl formation and reaction is common to many mononuclear ferrous enzymes, which participate in a broader range of reactions than their well-characterized counterparts, the haem enzymes. Here we report the first crystal structure of a 2-oxoacid-dependent oxygenase. High-resolution structures for apo-DAOCS, the enzyme complexed with Fe(II), and with Fe(II) and 2-oxoglutarate, were obtained from merohedrally twinned crystals. Using a model based on these structures, we propose a mechanism for ferryl formation.
The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and puri®ed by an ef®cient large-scale procedure. Puri®ed protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel ®ltration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized AEFe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A Ê resolution and belonged to the R3 space group (unit cell dimensions: a b 106.4 A Ê , c 71.2 A Ê ; a b 90 , g 120 (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry de®ned by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best ®t to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 A Ê , three nitrogen or oxygen atoms at 2.11 A Ê and one other light atom at 2.04 A Ê . For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A Ê ), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A Ê , another O atom at 2.08 A Ê and one at 2.03 A Ê . Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism.
Peroxisome proliferator-activated receptor gamma (PPARgamma) is well-known as the receptor of thiazolidinedione antidiabetic drugs. In this paper, we present a successful example of employing structure-based virtual screening, a method that combines shape-based database search with a docking study and analogue search, to discover a novel family of PPARgamma agonists based upon pyrazol-5-ylbenzenesulfonamide. Two analogues in the family show high affinity for, and specificity to, PPARgamma and act as partial agonists. They also demonstrate glucose-lowering efficacy in vivo. A structural biology study reveals that they both adopt a distinct binding mode and have no H-bonding interactions with PPARgamma. The absence of H-bonding interaction with the protein provides an explanation why both function as partial agonists since most full agonists form conserved H-bonds with the activation function helix (AF-2 helix) which, in turn, enhances the recruitment of coactivators. Moreover, the structural biology and computer docking studies reveal the specificity of the compounds for PPARgamma could be due to the restricted access to the binding pocket of other PPAR subtypes, i.e., PPARalpha and PPARdelta, and steric hindrance upon the ligand binding.
Deacetoxycephalosporin C synthase is an iron(II) 2-oxoglutaratedependent oxygenase that catalyzes the oxidative ring-expansion of penicillin N to deacetoxycephalosporin C. The wild-type enzyme is only able to efficiently utilize 2-oxoglutarate and 2-oxoadipate as a 2-oxoacid co-substrate. Mutation of arginine 258, the side chain of which forms an electrostatic interaction with the 5-carboxylate of the 2-oxoglutarate co-substrate, to a glutamine residue reduced activity to about 5% of the wild-type enzyme with 2-oxoglutarate. However, other aliphatic 2-oxoacids, which were not cosubstrates for the wild-type enzyme, were utilized by the R258Q mutant. These 2-oxoacids "rescued" catalytic activity to the level observed for the wild-type enzyme as judged by penicillin N and G conversion. These cosubstrates underwent oxidative decarboxylation as observed for 2-oxoglutarate in the normal reaction with the wild-type enzyme. Crystal structures of the iron(II)-2-oxo-3-methylbutanoate (1.5 Å), and iron(II)-2-oxo-4-methylpentanoate (1.6 Å) enzyme complexes were obtained, which reveal the molecular basis for this "chemical co-substrate rescue" and help to rationalize the co-substrate selectivity of 2-oxoglutaratedependent oxygenases. Deacetoxycephalosporin C synthase (DAOCS)1 is an iron(II) and 2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N to deacetoxycephalosporin C in the biosynthesis of cephem antibiotics in Streptomyces clavuligerus (1). The subsequent hydroxylation of DAOC to deacetylcephalosporin C (DAC) is catalyzed by a closely related enzyme, deacetylcephalosporin C synthase (DACS). In Cephalosporium acremonium a single, bifunctional protein, deacetoxy/deacetylcephalosporin C synthase (DAOC/DAC synthase), performs both reactions (2-4). S. clavuligerus also contains a 7␣-hydroxylase, which is involved in the biosynthesis of cephamycin C from DAC (5).DAOCS is a member of the iron(II) and 2-oxoglutarate-dependent oxygenase family, which catalyze a wide variety of oxidative reactions (6, 7). DAOCS, DACS, and DAOC/DAC synthase belong to a subgroup of more closely related enzymes, which have significant primary sequence homology to one another (6). Also included in this group are two enzymes which do not use 2-oxoglutarate as a co-substrate, isopenicillin N synthase (IPNS), the iron-dependent oxidase responsible for formation of the penicillin nucleus, and 1-amino-1-carboxycyclopropane oxidase (ACCO) which catalyzes the last step during ethylene biosynthesis in plants.Mechanistic understanding of iron(II), 2-oxoglutaratedependent oxygenases have been significantly advanced by the recently determined crystal structures of DAOCS (1,8,9) and clavaminic acid synthase (10). The DAOCS crystal structures revealed the presence of a number of arginine residues within the active site, with arginine 258 (part of a conserved RXS motif) being involved in co-substrate binding (1,8). The equivalent RXS motif residues in Aspergillus nidulans IPNS, arginine 281, and serine 283, bind the ␣-carboxylate gro...
Mitochondria are the powerhouses of cells. Mitochondrial CRaf is a potential cancer therapeutic target, as it regulates mitochondrial function and is localized to the mitochondria by its Nterminal domain. However, Raf inhibitor monotherapy can induce S338 phosphorylation of C-Raf (pC-Raf S338 ) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPK S308 ), which together became colocalized in the mitochondria to facilitate mitochondrial remodeling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anticancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knockout MEF C-RafÀ/À or siRNA knockdown ACHN renal cancer cells abrogated the cytotoxicity of combination therapy. Crystal structure simulation showed that GW5074 bound to C-Raf and induced a C-Raf conformational change that enhanced sorafenib-binding affinity.In the presence of pDAPK S308 , this drug-target interaction compromised the mitochondrial targeting effect of the N-terminal domain of C-Raf, which induced two-hit damages to cancer cells. First, combination therapy facilitated pC-Raf S338 and pDAPK S308 translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and reactive oxygen species (ROS) generation. Second, ROS facilitated PP2A-mediated dephosphorylation of pDAPK S308 to DAPK. PP2A then dissociated from the C-Raf-DAPK complex and induced profound cancer cell death. Increased pDAPK S308 modification was also observed in renal cancer tissues, which correlated with poor disease-free survival and poor overall survival in renal cancer patients. Besides mediating the anticancer effect, pDAPK S308 may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation. Cancer Res; 75(17); 3568-82. Ó2015 AACR.
Human 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) is a putative iron-containing non-heme oxygenase of unknown specificity and biological significance. We report 25 families containing 34 individuals with neurological disease associated with biallelic HPDL variants. Phenotypes ranged from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spasticity and global developmental delays, sometimes complicated by episodes of neurological and respiratory decompensation. Variants included bona fide pathogenic truncating changes, although most were missense substitutions. Functionality of variants could not be determined directly as the enzymatic specificity of HPDL is unknown; however, when HPDL missense substitutions were introduced into 4-hydroxyphenylpyruvate dioxygenase (HPPD, an HPDL orthologue), they impaired the ability of HPPD to convert 4-hydroxyphenylpyruvate into homogentisate. Moreover, three additional sets of experiments provided evidence for a role of HPDL in the nervous system and further supported its link to neurological disease: (i) HPDL was expressed in the nervous system and expression increased during neural differentiation; (ii) knockdown of zebrafish hpdl led to abnormal motor behaviour, replicating aspects of the human disease; and (iii) HPDL localized to mitochondria, consistent with mitochondrial disease that is often associated with neurological manifestations. Our findings suggest that biallelic HPDL variants cause a syndrome varying from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spastic tetraplegia associated with global developmental delays.
Deacetoxycephalosporin C synthase (DAOCS) catalyses the oxidative ring expansion of penicillin N, the committed step in the biosynthesis of cephamycin C by Streptomyces clavuligerus. Site-directed mutagenesis was used to investigate the seven Arg residues for activity (74, 75, 160, 162, 266, 306 and 307), selected on the basis of the DAOCS crystal structure. Greater than 95% of activity was lost upon mutation of Arg-160 and Arg266 to glutamine or other residues. These results are consistent with the proposed roles for these residues in binding the carboxylate linked to the nucleus of penicillin N (Arg160 and Arg162) and the carboxylate of the a-aminoadipoyl side-chain (Arg266). The results for mutation of Arg74 and Arg75 indicate that these residues play a less important role in catalysis/binding. Together with previous work, the mutation results for Arg306 and Arg307 indicate that modification of the C-terminus may be profitable with respect to altering the penicillin side-chain selectivity of DAOCS.
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