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We present the synthesis and coordination chemistry of a bulky, tripodal N,N,O ligand, ImPh2NNO tBu (L), designed to model the 2‐His‐1‐carboxylate facial triad (2H1C) by means of two imidazole groups and an anionic 2,4‐di‐tert‐butyl‐subtituted phenolate. Reacting K‐L with MCl2 (M = Fe, Zn) affords the isostructural, tetrahedral non‐heme complexes [Fe(L)(Cl)] (1) and [Zn(L)(Cl)] (2) in high yield. The tridentate N,N,O ligand coordination observed in their X‐ray crystal structures remains intact and well‐defined in MeCN and CH2Cl2 solution. Reacting 2 with NaSPh affords a tetrahedral zinc thiolate complex, [Zn(L)(SPh)] (4), that is relevant to isopenicillin N synthase (IPNS) biomimicry. Cyclic voltammetry studies demonstrate the ligand's redox non‐innocence, where phenolate oxidation is the first electrochemical response observed in K‐L, 2 and 4. However, the first electrochemical oxidation in 1 is iron‐centred, the assignment of which is supported by DFT calculations. Overall, ImPh2NNO tBu provides access to well‐defined mononuclear, monoligated, N,N,O‐bound metal complexes, enabling more accurate structural modelling of the 2H1C to be achieved., How N,N,O can you go? A new bulky, tripodal N,N,O phenolate ligand provides access to well‐defined mononuclear, monoligated non‐heme iron and zinc complexes that accurately model the 2‐His‐1‐carboxylate facial triad. These complexes are ideal synthons from which to create a range of biomimetic N,N,O‐bound complexes featuring biorelevant cofactors.

Metal sites in biology catalyse some of the most challenging and consequential reactions on Earth, ranging from oxygen transport in animals to photosynthesis in plants and nitrogen-fixation in bacteria. This astounding breadth of reactivity includes attractive features such as small molecule activation and late-stage functionalisation, all of which are achieved under physiological conditions and with exquisite stereo- and regioselectivity. For bioinorganic chemists, the direct deployment, engineering or mimicry of metalloenzymes and metal-containing biomolecules in research and commercial applications holds great potential for improving the green credentials of today’s chemical industry. The present thesis describes the synthesis, structure and reactivity of bioinspired iron complexes that model O2-activating non-heme iron enzymes such as isopenicillin N synthase (IPNS), which contains the 2-His-1-Carboxylate facial triad (2H1C) at its active site. A family of bioinspired N,N,O phenolate ligands was developed to structurally model the 2H1C and their coordination chemistry to iron and zinc was explored. These efforts resulted in the creation of an interesting series of N,N,O-bound iron and zinc thiolate complexes that model important structural aspects of the substrate-bound active site of IPNS. This research also showed that mononuclear N,N,O-bound iron(II) alpha-ketoacid complexes could be synthesised, opening up a new field of exploration related to alpha-ketoglutarate-dependent enzymes, the largest sub-family of 2H1C-containing non-heme iron enzymes.

Non-heme iron complexes with cis-FeIII(OH)(SAr/OAr) coordination were isolated and examined for their reactivity with a tertiary carbon radical. The sulfur-ligated complex shows a temperature dependence on •OH versus ArS• transfer, whereas the oxygen-ligated complex does not. These results provide the first working model for C-S bond formation in isopenicillin N synthase and indicate that kinetic control may be a key factor in the selectivity of non-heme iron "rebound" processes.

An effective and simple-to-implement approach for anaerobic room-temperature data collection is described and demonstrated by application to dioxygen utilizing enzymes., Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitro­gen cryo-stream at 100 K) enable, is data collection of di­oxy­gen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for di­oxy­gen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the ‘sheet-on-sheet’ sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (

Complementary XFEL methods reveal O2 reaction intermediates and global protein dynamics during Isopenicillin N synthase catalysis., Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.

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.

Transport of penicillin intermediates and penicillin secretion are still poorly characterized in Penicillium chrysogenum (re-identified as Penicillium rubens ). Calcium (Ca 2 + ) plays an important role in the metabolism of filamentous fungi, and casein phosphopeptides (CPP) are involved in Ca 2 + internalization. In this study we observe that the effect of CaCl 2 and CPP is additive and promotes an increase in penicillin production of up to 10–12 fold. Combination of CaCl 2 and CPP greatly promotes expression of the three penicillin biosynthetic genes. Comparative proteomic analysis by 2D-DIGE, identified 39 proteins differentially represented in P. chrysogenum Wisconsin 54-1255 after CPP/CaCl 2 addition. The most interesting group of overrepresented proteins were a peroxisomal catalase, three proteins of the methylcitrate cycle, two aminotransferases and cystationine β-synthase, which are directly or indirectly related to the formation of penicillin amino acid precursors. Importantly, two of the enzymes of the penicillin pathway (isopenicillin N synthase and isopenicillin N acyltransferase) are clearly induced after CPP/CaCl 2 addition. Most of these overrepresented proteins are either authentic peroxisomal proteins or microbody-associated proteins. This evidence suggests that addition of CPP/CaCl 2 promotes the formation of penicillin precursors and the penicillin biosynthetic enzymes in peroxisomes and vesicles, which may be involved in transport and secretion of penicillin. Significance Penicillin biosynthesis in Penicillium chrysogenum is one of the best characterized secondary metabolism processes. However, the mechanism by which penicillin is secreted still remains to be elucidated. Taking into account the role played by Ca 2 + and CPP in the secretory pathway and considering the positive effect that Ca 2 + exerts on penicillin production, the analysis of global protein changes produced after CPP/CaCl 2 addition is very helpful to decipher the processes related to the biosynthesis and secretion of penicillin.

Isopenicillin N synthase (IPNS) is a nonheme-Fe2+-dependent enzyme that mediates a key step in penicillin biosynthesis. It catalyses the conversion of the tripeptide δ-( l -α-aminoadipoyl)- l -cysteine- d -valine (ACV) to isopenicillin N, which is a key precursor to β-lactam antibiotics. The pa4191 gene in Pseudomonas aeruginosa PAO1 has provisionally been annotated as a member of the IPNS family. In this work, we report the crystal structure of PA4191 from P. aeruginosa (PaIPNS hereafter). The 1.65 A resolution PaIPNS structure forms a jelly roll fold and is confirmed to be a member of the IPNS family based on structural homology. A metal centre within the jelly roll consists of the strictly conserved His201, Asp203 and His257 residues. MicroScale Thermophoresis binding analysis confirms that PaIPNS is a metal-binding protein with a strong preference for iron, but that it does not bind the tripeptide ACV. Structural comparison of PaIPNS with a previously reported IPNS-ACV complex structure reveals a restricted binding pocket that is unable to accommodate ACV.

This review discusses the current mechanistic understanding of a group of mononuclear non-heme iron-dependent enzymes that catalyze four-electron oxidation of their organic substrates without the use of any cofactors or cosubstrates. One set of enzymes acts on α-ketoacid-containing substrates, coupling decarboxylation to oxygen activation. This group includes 4-hydroxyphenylpyruvate dioxygenase, 4-hydroxymandelate synthase, and CloR involved in clorobiocin biosynthesis. A second set of enzymes acts on substrates containing a thiol group that coordinates to the iron. This group is comprised of isopenicillin N synthase, thiol dioxygenases, and enzymes involved in the biosynthesis of ergothioneine and ovothiol. The final group of enzymes includes HEPD and MPnS that both carry out the oxidative cleavage of the carbon-carbon bond of 2-hydroxyethylphosphonate but generate different products. Commonalities amongst many of these enzymes are discussed and include the initial substrate oxidation by a ferric-superoxo-intermediate and a second oxidation by a ferryl species.

The enzyme isopenicillin N synthase (IPNS) installs the β-lactam and thiazolidine rings of the penicillin core into the linear tripeptide, L-δ-aminoadipoyl-L-Cys-D-Val (ACV), on the pathways to a number of important antibacterial drugs. A classic set of enzymological and crystallographic studies by Baldwin and co-workers established that this overall four-electron oxidation occurs by a sequence of two oxidative cyclizations, with the β-lactam ring being installed first and the thiazolidine ring second. Each phase requires cleavage of an aliphatic C–H bond of the substrate: the pro-S-CCys,β-H bond for closure of the β-lactam ring, and the CVal,β-H bond for installation of the thiazolidine ring. IPNS uses a mononuclear non-heme-iron(II) cofactor and dioxygen as co-substrate to cleave these C–H bonds and direct the ring closures. Despite the intense scrutiny to which the enzyme has been subjected, the identities of the oxidized iron intermediates that cleave the C–H bonds have been addressed only computationally; no experimental insight into their geometric or electronic structures has been reported. In this work, we have employed a combination of transient-state-kinetic and spectroscopic methods, together with the specifically deuterium-labeled substrates, A[d2-C]V and AC[d8-V], to identify both C–H-cleaving intermediates. The results show that they are high-spin Fe(III)-superoxo and high-spin Fe(IV)-oxo complexes, respectively, in agreement with published mechanistic proposals derived computationally from Baldwin’s founding work.

Dioxygen activation by FeII thiolate complexes is relatively rare in biological and chemical systems because the sulfur site is at least as vulnerable as the iron site to oxidative modification. O2 activation by FeII-SR complexes with thiolate bound trans to the O2 binding site generally affords the FeIV[double bond, length as m-dash]O intermediate and oxidized thiolate. On the other hand, O2 activation by Fe(ii)-SR complexes with thiolate bound cis to the O2 binding site generates FeIII-O-FeIII or S-oxygenated complexes. The postulated FeIV[double bond, length as m-dash]O intermediate has only been identified in isopenicillin N synthase recently. We demonstrated here that O2 activation by a dinuclear FeII thiolate-rich complex produces a mononuclear FeIII complex and water with a supply of electron donors. The thiolate is bound cis to the postulated dioxygen binding site, and no FeIII-O-FeIII or S-oxygenated complex was observed. Although we have not detected the transient intermediate by spectroscopic measurements, the FeIV[double bond, length as m-dash]O intermediate is suggested to exist by theoretical calculation, and P-oxidation and hydride-transfer experiments. In addition, an unprecedented FeIII-O2-FeIII complex supported by thiolates was observed during the reaction by using a coldspray ionization time-of-flight mass (CSI-TOF MS) instrument. This is also supported by low-temperature UV-vis measurements. The intramolecular NHO[double bond, length as m-dash]FeIV hydrogen bonding, calculated by DFT, probably fine tunes the O2-activation process for intramolecular hydrogen abstraction, avoiding the S-oxygenation at cis-thiolate.

Background Autophagy is used for degradation of cellular components and nutrient recycling. Atg8 is one of the core proteins in autophagy and used as a marker for autophagic detection. However, the autophagy of filamentous fungi is poorly understood compared with that of Saccharomyces cerevisiae. Our previous study revealed that disruption of the autophagy related gene Acatg1 significantly enhanced cephalosporin C yield through reducing degradation of cephalosporin biosynthetic proteins in Acremonium chrysogenum, suggesting that modulation of autophagic process is one promising way to increase antibiotic production in A. chrysogenum. Results In this study, a S. cerevisiae ATG8 homologue gene Acatg8 was identified from A. chrysogenum. Acatg8 could complement the ATG8 mutation in S. cerevisiae, indicating that Acatg8 is a functional homologue of ATG8. Microscope observation demonstrated the fluorescently labeled AcAtg8 was localized in the cytoplasm and autophagosome of A. chrysogenum, and the expression of Acatg8 was induced by nutrient starvation. Gene disruption and genetic complementation revealed that Acatg8 is essential for autophagosome formation. Disruption of Acatg8 significantly reduced fungal conidiation and delayed conidial germination. Localization of GFP-AcAtg8 implied that autophagy is involved in the early phase of conidial germination. Similar to Acatg1, disruption of Acatg8 remarkably enhanced cephalosporin C yield. The cephalosporin C biosynthetic enzymes (isopenicillin N synthase PcbC and isopenicillin N epimerase CefD2) and peroxisomes were accumulated in the Acatg8 disruption mutant (∆Acatg8), which might be the main reasons for the enhancement of cephalosporin C production. However, the biomass of ΔAcatg8 decreased drastically at the late stage of fermentation, suggesting that autophagy is critical for A. chrysogenum cell survival under nutrition deprived condition. Disruption of Acatg8 also resulted in accumulation of mitochondria, which might produce more reactive oxygen species (ROS) which promotes fungal death. However, the premature death is unfavorable for cephalosporin C production. To solve this problem, a plasmid containing Acatg8 under control of the xylose/xylan-inducible promoter was introduced into ∆Acatg8. Conidiation and growth of the recombinant strain restored to the wild-type level in the medium supplemented with xylose, while the cephalosporin C production maintained at a high level even prolonged fermentation. Conclusions Our results demonstrated inducible expression of Acatg8 and disruption of Acatg8 remarkably increased cephalosporin C production. This study provides a promising approach for yield improvement of cephalosporin C in A. chrysogenum. Electronic supplementary material The online version of this article (10.1186/s12934-018-1021-9) contains supplementary material, which is available to authorized users.

The Rieske dioxygenases are a major subclass of mononuclear nonheme iron enzymes that play an important role in bioremediation. Recently, a high-spin FeIII–(hydro)-peroxy intermediate (BZDOp) has been trapped in the peroxide shunt reaction of benzoate 1,2-dioxygenase. Defining the structure of this intermediate is essential to understanding the reactivity of these enzymes. Nuclear resonance vibrational spectroscopy (NRVS) is a recently developed synchrotron technique that is ideal for obtaining vibrational, and thus structural, information on Fe sites, as it gives complete information on all vibrational normal modes containing Fe displacement. In this study, we present NRVS data on BZDOp and assign its structure using these data coupled to experimentally calibrated density functional theory calculations. From this NRVS structure, we define the mechanism for the peroxide shunt reaction. The relevance of the peroxide shunt to the native FeII/O2 reaction is evaluated. For the native FeII/O2 reaction, an FeIII–superoxo intermediate is found to react directly with substrate. This process, while uphill thermodynamically, is found to be driven by the highly favorable thermodynamics of proton-coupled electron transfer with an electron provided by the Rieske [2Fe-2S] center at a later step in the reaction. These results offer important insight into the relative reactivities of FeIII–superoxo and FeIII–hydroperoxo species in nonheme Fe biochemistry.

International audience; Oxoiron(IV) species are implicated as reactive intermediates in nonheme monoiron oxygenases, often acting as the agent for hydrogen-atom transfer from substrate. A histidine is the most likely ligand trans to the oxo unit in most enzymes characterized thus far but is replaced by a carboxylate in the case of isopenicillin N synthase. As the effect of a trans carboxylate ligand on the properties of the oxoiron(IV) unit has not been systematically studied, we have synthesized and characterized four oxoiron(IV) complexes supported by the tetramethylcyclam (TMC) macrocycle and having a carboxylate ligand trans to the oxo unit. Two complexes have acetate or propionate axial ligands, while the other two have the carboxylate functionality tethered to the macrocyclic ligand framework by one or two methylene units. Interestingly, these four complexes exhibit substrate oxidation rates that differ by more than 100-fold, despite having Ep,c values for the reduction of the Fe═O unit that span a range of only 130 mV. Eyring parameters for 1,4-cyclohexadiene oxidation show that reactivity differences originate from differences in activation enthalpy between complexes with tethered carboxylates and those with untethered carboxylates, in agreement with computational results. As noted previously for the initial subset of four complexes, the logarithms of the oxygen atom transfer rates of 11 complexes of the FeIV(O)TMC(X) series increase linearly with the observed Ep,c values, reflecting the electrophilicity of the Fe═O unit. In contrast, no correlation with Ep,c values is observed for the corresponding hydrogen atom transfer (HAT) reaction rates; instead, the HAT rates increase as the computed triplet–quintet spin state gap narrows, consistent with Shaik’s two-state-reactivity model. In fact, the two complexes with untethered carboxylates are among the most reactive HAT agents in this series, demonstrating that the axial ligand can play a key role in tuning the HAT reactivity in a nonheme iron enzyme active site.

The machinery of antibiotic production by Penicillium chrysogenum PQ-96 is composed of co-located cytosolic and peroxisomal enzymes of the penicillin G biosynthesis pathway. Pexophagy and exocytosis should be currently considered as an alternative for penicillin G secretion from the mycelial cells. Penicillin G overproduction is a cellular detoxification process, protecting the mycelium from the toxicity of the antibiotic precursor. 1. Introduction. 2. Peroxisomal functions and penicillin G biosynthesis. 3. Immunoelectron microscopyof isopenicillin N synthase. 4. Ultrastructural localization of peroxisomes. 5. Pexophagy and exocytosis – secretion of penicillin G. 6. Conclusions

Describing the organization of substrates and substrate analogues in the active site of cysteine dioxygenase identifies potential intermediates in this critical yet poorly understood reaction, the oxidation of cysteine to cysteine sulfinic acid. The fortuitous formation of persulfides under crystallization conditions has allowed their binding in the active site of cysteine dioxygenase to be studied. The crystal structures of cysteine persulfide and 3-mercaptopropionic acid persulfide bound to iron(II) in the active site show that binding of the persulfide occurs via the distal sulfide and, in the case of the cysteine persulfide, the amine also binds. Persulfide was detected by mass spectrometry in both the crystal and the drop, suggesting its origin is chemical rather than enzymatic. A mechanism involving the formation of the relevant disulfide from sulfide produced by hydrolysis of dithionite is proposed. In comparison, persulfenate {observed bound to cysteine dioxygenase [Simmons, C. R., et al. (2008) Biochemistry 47, 11390]} is shown through mass spectrometry to occur only in the crystal and not in the surrounding drop, suggesting that in the crystalline state the persulfenate does not lie on the reaction pathway. Stabilization of both the persulfenate and the persulfides does, however, suggest the position in which dioxygen binds during catalysis.

Isopenicillin N synthase (IPNS) is a non-heme iron oxidase central to the biosynthesis of β-lactam antibiotics. IPNS converts the tripeptide δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to isopenicillin N while reducing molecular oxygen to water. The substrate analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-O-methyl-d-threonine (ACmT) is not turned over by IPNS. Epimeric δ-(l-α-aminoadipoyl)-l-cysteinyl-O-methyl-d-allo-threonine (ACmaT) is converted to a bioactive penam product. ACmT and ACmaT differ from each other only in the stereochemistry at the β-carbon atom of their third residue. These substrates both contain a methyl ether in place of the isopropyl group of ACV. We report an X-ray crystal structure for the anaerobic IPNS:Fe(II):ACmT complex. This structure reveals an additional water molecule bound to the active site metal, held by hydrogen-bonding to the ether oxygen atom of the substrate analogue.

Genetic engineering of fungal cell factories mainly focuses on manipulating enzymes of the product pathway or primary metabolism. However, despite the use of strong promoters or strains containing the genes of interest in multiple copies, the desired strongly enhanced enzyme levels are often not obtained.Here we present a novel strategy to improve penicillin biosynthesis by Penicillium chrysogenum by reducing reactive and toxic metabolic by-products, 2-oxoaldehydes. This was achieved by overexpressing the genes encoding glyoxalase I and II, which resulted in a 10% increase in penicillin titers relative to the control strain.The protein levels of two key enzymes of penicillin biosynthesis, isopenicillin N synthase and isopenicillin N acyltransferase, were increased in the glyoxalase transformants, whereas their transcript levels remained unaltered. These results suggest that directed intracellular reduction of 2-oxoaldehydes prolongs the functional lifetime of these enzymes. (C) 2013 Elsevier Inc. All rights reserved.

Beta-lactam biosynthesis was thought to occur only in fungi and bacteria, but we recently reported the presence of isopenicillin N synthase in a soil-dwelling animal, Folsomia candida. However, it has remained unclear whether this gene is part of a larger beta-lactam biosynthesis pathway and how widespread the occurrence of penicillin biosynthesis is among animals. Here, we analysed the distribution of beta-lactam biosynthesis genes throughout the animal kingdom and identified a beta-lactam gene cluster in the genome of F. candida (Collembola), consisting of isopenicillin N synthase (IPNS), δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS), and two cephamycin C genes (cmcI and cmcJ) on a genomic scaffold of 0.76 Mb. All genes are transcriptionally active and are inducible by stress (heat shock). A beta-lactam compound was detected in vivo using an ELISA beta-lactam assay. The gene cluster also contains an ABC transporter which is coregulated with IPNS and ACVS after heat shock. Furthermore, we show that different combinations of beta-lactam biosynthesis genes are present in over 60% of springtail families, but they are absent from genome- and transcript libraries of other animals including close relatives of springtails (Protura, Diplura and insects). The presence of beta-lactam genes is strongly correlated with an euedaphic (soil-living) lifestyle. Beta-lactam genes IPNS and ACVS each form a phylogenetic clade in between bacteria and fungi, while cmcI and cmcJ genes cluster within bacteria. This suggests a single horizontal gene transfer event most probably from a bacterial host, followed by differential loss in more recently evolving species.

The non-heme ferrous dependent oxidase isopenicillin N synthase (IPNS) catalyses the biosynthesis of isopenicillin N from a tripeptide substrate. The crystal structure of Aspergillus nidulans IPNS complexed to manganese reveals a six co-ordinate metal ligated by two water molecules and four protein ligands: His-214, His-270, Asp-216 and Gln-330 (the penultimate C-terminal residue). Modification of Gln-330 to Ala or Leu, or deletion of 2 or 6 residues from the C-terminus resulted in lowering of specific activity; no activity was observed after deletion of 8 residues. The results demonstrate that metal ligation by Gln-330 is not required for catalytic activity.© 1997 Federation of European Biochemical Societies.

The role of the L-δ-(α-aminoadipoyl)–Lcys teinyl amide bond in isopenicillin N synthase catalysis is probed by the synthesis of two analogues of its tripeptide substrate L-δ-α-aminoadipoyl-L-cysteinyl- D-valine.

Isopenicillin N synthase (IPNS) converts the linear tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) into bicyclic isopenicillin N (IPN) in the central step in the biosynthesis of penicillin and cephalosporin antibiotics. Solution-phase incubation experiments have shown that IPNS turns over analogues with a diverse range of side chains in the third (valinyl) position of the substrate, but copes less well with changes in the second (cysteinyl) residue. IPNS thus converts the homologated tripeptides δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-valine (AhCV) and δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-allylglycine (AhCaG) into monocyclic hydroxy-lactam products; this suggests that the additional methylene unit in these substrates induces conformational changes that preclude second ring closure after initial lactam formation. To investigate this and solution-phase results with other tripeptides δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-Xaa, we have crystallised AhCV and δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-S-methylcysteine (AhCmC) with IPNS and solved crystal structures for the resulting complexes. The IPNS:Fe(II):AhCV complex shows diffuse electron density for several regions of the substrate, revealing considerable conformational freedom within the active site. The substrate is more clearly resolved in the IPNS:Fe(II):AhCmC complex, by virtue of thioether coordination to iron. AhCmC occupies two distinct conformations, both distorted relative to the natural substrate ACV, in order to accommodate the extra methylene group in the second residue. Attempts to turn these substrates over within crystalline IPNS using hyperbaric oxygenation give rise to product mixtures.

The final step in the biosynthesis of the plant signaling molecule ethylene is catalyzed by 1-aminocyclopropane-1-carboxylate (ACC) oxidase, a member of the non-heme iron(II) dependent family of oxygenases and oxidases, which has a requirement for ascorbate as a co-substrate and carbon dioxide as an activator. ACC oxidase (tomato) has a particularly short half-life under catalytic conditions undergoing metal-catalyzed oxidative (MCO) fragmentation. Sequence comparisons of ACC oxidases with isopenicillin N synthase (IPNS) and members of the 2-oxoglutarate Fe(II) dependent dioxygenases show an aspartate and two of six ACC oxidase conserved histidine residues are completely conserved throughout this subfamily of Fe(II) dependent oxygenases/oxidases. Previous mutagenesis, spectroscopic, and crystallographic studies on IPNS indicate that the two completely conserved histidine and aspartate residues act as Fe(II) ligands. To investigate the role of the conserved aspartate and histidine residues in ACC oxidase (tomato fruit), they were substituted via site-directed mutagenesis. Modified ACC oxidases produced were H39Q, H56Q, H94Q, H177Q, H177D, H177E, D179E, D179N, H177DandD179E, H211Q, H234Q, H234D, and H234E. Among those histidine mutants replaced by glutamine, H39Q, H56Q, H94Q, and H211Q were catalytically active, indicating these histidines are not essential for catalysis. Mutant enzymes H177D, H177Q, D179N, H177DandD179E, H234Q, H234D, and H234E were catalytically inactive consistent with the assignment of H177, D179, and H234 as iron ligands. Replacement of H177 with glutamate or D179 with glutamate resulted in modified ACC oxidases which still effected the conversion of ACC to ethylene, albeit at a very low level of activity, which was stimulated by bicarbonate. The H177D (inactive), H177E (low activity), D179E (low activity), and H234Q (inactive) modified ACC oxidases all underwent MCO fragmentation, indicating that they can bind iron, dioxygen, ACC, and ascorbate. The results suggest that MCO cleavage results from active site-mediated reactions and imply that, while H177, D179, and H234 are all involved in metal ligation during catalysis, ligation to H234 is not required for fragmentation. It is possible that MCO fragmentation results from reaction of incorrectly folded or "primed" ACC oxidase.

Isopenicillin N synthase (IPNS) was cocrystallised with ferrous sulphate and its substrate, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (Aad-Cys-Val). Vital to the successful procedure was the maintenance of a rigorously anaerobic environment. Hanging-drop vapour-diffusion crystallisation experiments, using lithium sulphate as the precipitant produced three crystal forms. Form I crystals, with a plate habit, diffracted X-rays to at least 0.11-nm resolution at the European Synchrotron Radiation Facility and belong to the space group P2(1)2(1)2(1), with unit-cell dimensions a = 4.68, b = 7.15, c = 10.10 nm. Their asymmetric unit contains a single IPNS.Fe(II).Aad-Cys-Val complex with a solvent content of 38.5%. Form II crystals, with a hexagonal habit, diffract X-rays to at least 0.21 nm resolution at the European Synchrotron Radiation Facility and belong to the space group P3(1)21, with unit-cell dimensions a = 10.10, b = 10.10, c = 11.567 nm. Their asymmetric unit also contains a single IPNS.Fe(II).Aad-Cys-Val complex with a solvent content of 69.5%. Form III crystals, needles, do not show well-ordered diffraction. Although all three forms were initially produced in crystallisation experiments under identical conditions, appropriate micro and streak seeding allows selective crystallisation of form I or form II crystals. Extended X-ray-absorption fine-structure studies on a crystalline slurry of the form I crystals demonstrate the presence of an Fe-S(Aad-Cys-Val) bond length of 0.234 +/- 0.003 nm.

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.

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.

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 Å 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.

Intense classical strain improvement has yielded industrial Penicillium chrysogenum strains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)- l -cysteinyl- d -valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme.

Industrial penicillin production levels by the filamentous fungus Penicillium chrysogenum increased dramatically by classical strain improvement. High-yielding strains contain multiple copies of the penicillin biosynthetic gene cluster that encodes three key enzymes of the β-lactam biosynthetic pathway. We have analyzed the gene cluster dose effect on penicillin production using the high-yielding P. chrysogenum strain DS17690 that was cured from its native clusters. The amount of penicillin V produced increased with the penicillin biosynthetic gene cluster number but was saturated at high copy numbers. Likewise, transcript levels of the biosynthetic genes pcbAB [δ-( l -α-aminoadipyl)- l -cysteinyl- d -valine synthetase], pcbC (isopenicillin N synthase), and penDE (acyltransferase) correlated with the cluster copy number. Remarkably, the protein level of acyltransferase, which localizes to peroxisomes, was saturated already at low cluster copy numbers. At higher copy numbers, intracellular levels of isopenicillin N increased, suggesting that the acyltransferase reaction presents a limiting step at a high gene dose. Since the number and appearance of the peroxisomes did not change significantly with the gene cluster copy number, we conclude that the acyltransferase activity is limiting for penicillin biosynthesis at high biosynthetic gene cluster copy numbers. These results suggest that at a high penicillin production level, productivity is limited by the peroxisomal acyltransferase import activity and/or the availability of coenzyme A (CoA)-activated side chains.

In this study, the effects of glycerol on cephalosporin C production by Acremonium chrysogenum M35 were evaluated. The addition of glycerol increased cephalosporin production by up to 12-fold. Glycerol caused the upregulation of the transcription of the isopenicillin synthase (pcbC) and transporter (cefT) genes in early exponential phase, and affected the cell morphology since hyphal fragments differentiated into arthrospores. These results indicate that glycerol effectively enhances cephalosporin C production via stimulation of cell differentiation.

Isopenicillin N synthase (IPNS) can have both oxidase and oxygenase activity depending on the substrate. For the native substrate, ACV, oxidase activity exists; however, for the substrate analogue ACOV, which lacks an amide nitrogen, IPNS exhibits oxygenase activity. The potential energy surfaces for the O-O bond elongation and cleavage were calculated for three different reactions: homolytic cleavage via traditional Fenton chemistry, heterolytic cleavage, and nucleophilic attack. These surfaces show that the hydroperoxide-ferrous intermediate, formed by O(2)-activated H atom abstraction from the substrate, can exploit different reaction pathways and that interactions with the substrate govern the pathway. The hydrogen bonds from hydroperoxide to the amide nitrogen of ACV polarize the sigma* orbital of the peroxide toward the proximal oxygen, facilitating heterolytic cleavage. For the substrate analogue ACOV, this hydrogen bond is no longer present, leading to nucleophilic attack on the substrate intermediate C-S bond. After cleavage of the hydroperoxide, the two reaction pathways proceed with minimal barriers, resulting in the closure of the beta-lactam ring for the oxidase activity (ACV) or formation of the thiocarboxylate for oxygenase activity (ACOV).

Isopenicillin N synthase (IPNS) catalyzes a key step in the biosynthesis of the important beta-lactam antibiotics penicillins and cephalosporins. Density-functional calculations with the B3LYP functional are used to propose a detailed mechanism for this reaction. The results support the general scheme outlined from experimental observations, with formation of a four-membered beta-lactam ring followed by formation of a five-membered thiazolidine ring. However, an alternative mechanism for the heterolytic O-O bond cleavage and beta-lactam ring formation steps is proposed. The former part involves protonation of the distal oxygen by an iron-bound water ligand. This mechanism highlights the strong similarities that exist between IPNS and other enzymes of the 2-histidine-1-carboxylate family, especially pterin-dependent amino acid hydroxylases and alpha-keto acid-dependent dioxygenases. Both activation of the cysteine beta-C-H bond by an iron-bound superoxo radical and activation of the valine beta-C-H bond by a ferryl-oxo species show reaction barriers close to the experimentally measured one. These results are in agreement with kinetic isotope experiments that suggest both C-H bond activation steps to be partially rate limiting. The ring formation sequence is determined by the relative strengths of the two C-H bonds. Only the ferryl-oxo intermediate is capable of activating the stronger valine beta-C-H bond.

Isopenicillin N synthase (IPNS), a non-heme iron oxidase central to penicillin and cephalosporin biosynthesis, catalyzes an energetically demanding chemical transformation to produce isopenicillin N from the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV). We describe the synthesis of two cyclopropyl-containing tripeptide analogues, delta-(l-alpha-aminoadipoyl)-l-cysteinyl-beta-methyl-d-cyclopropylglycine and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-cyclopropylglycine, designed as probes for the mechanism of IPNS. We have solved the X-ray crystal structures of these substrates in complex with IPNS and propose a revised mechanism for the IPNS-mediated turnover of these compounds. Relative to the previously determined IPNS-Fe(II)-ACV structure, key differences exist in substrate orientation and water occupancy, which allow for an explanation of the differences in reactivity of these substrates.

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum , strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC , encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.

This chapter summarizes the diverse array of biochemical transformations that are catalysed by Fe(ii)- and 2-oxoglutarate (2OG)-dependent oxygenases. One group of these enzymes utilizes protein substrates and functions in structural stabilization, oxygen sensing, histone-dependent regulation, or other roles. A second set of 2OG-dependent oxygenases acts on polynucleotides with functions that include DNA/RNA repair, regulation of transcription, biosynthesis of unique bases, and demethylation of 5-methylcytosine. A third assemblage of enzymes in this family is involved in lipid-related metabolism and function in carnitine biosynthesis, degradation of phytanic acids, and modification of various lipids. A fourth collection of these oxygenases catalyses reactions related to synthesis of flavonoids, anthocyanins, gibberellins, alkaloids and other metabolites found predominantly in plants. A fifth group of these enzymes acts on a variety of small molecules including free amino acids, nucleobases/nucleosides, herbicides, sulfonates/sulfates and phosphonates. A sixth compilation of 2OG-dependent oxygenases is utilized for antibiotic biosynthesis, including several halogenating enzymes. Finally, a seventh set of these enzymes is related in structure or mechanism to the 2OG-dependent oxygenases, but do not utilize 2OG, and include isopenicillin N synthase, a plant-specific ethylene-forming enzyme, and two enzymes that use 4-hydroxyphenylpyruvate. This introduction to the biochemical diversity of these amazing enzymes provides a foundation for appreciating the specific aspects detailed in the remaining chapters of this text.

Isopenicillin N synthase (IPNS) catalyses the oxidative bicyclization of δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to form isopenicillin N (IPN), a conversion that is essential in the biosynthesis of penicillin and cephalosporin antibiotics. IPNS uses the full oxidizing potential of molecular oxygen to drive this reaction, forming new C–N and C–S bonds and reducing O2 to two molecules of water. IPNS does not use 2-oxoglutarate (2OG) or any co-substrate other than O2, but has sequence homology along with structural and mechanistic similarities to the 2OG-dependent oxygenase family. The chemically challenging reaction that IPNS catalyses and the central role it plays in antibiotic biosynthesis mean that IPNS has been studied extensively over many years. Crystal structures for the substrate-free enzyme, enzyme–substrate and enzyme–product complexes have all been described, and key intermediates have been trapped chemically and characterized crystallographically as well. This structural information combines with a diverse array of solution phase turnover experiments and spectroscopic studies to provide a detailed picture of IPNS catalysis.