Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in β-lactam biosynthesis

Rabe, Patrick; Kamps, Jos J. A. G.; Schofield, Christopher J.; Lohans, Christopher T.

doi:10.1039/c8np00002f

Cited by 34 publications

(30 citation statements)

References 182 publications

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“…The majority of microbial natural products share a common biosynthetic logic and can be assigned to just a handful of major classes, including terpenes, saccharides, polyketides, alkaloids, and peptides . Natural products are synthesized through complex secondary metabolic pathways consisting of enzymes that can catalyze challenging sequential chemical transformations in a regioselective and stereoselective manner . Microbial natural product biosynthetic pathways are typically organized in self‐contained gene clusters that can be over 100 kb in length and encode over 40 biosynthetic enzymes .…”

Section: Natural Product Biosynthesis and Comparative Genomicsmentioning

confidence: 99%

“…Enzymes of the 2‐oxoglutarate (2OG) and nonhaem iron‐dependent oxygenases are able to perform energetically demanding chemical transformations and are common components of secondary metabolic pathways . Their functional divergence is particularly prevalent in the formation of β‐lactams, where the enzymes catalyze various hydroxylation, cyclization, desaturation, and epimerization reactions . Studies on fungal meroterpenoids (Fig.…”

Section: Direct Modulation Of Enzyme Function In Biosynthetic Gene CLmentioning

confidence: 99%

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A pharmaceutical model for the molecular evolution of microbial natural products

Fewer

Metsä‐Ketelä

2019

The FEBS Journal

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Microbes are talented chemists with the ability to generate tremendously complex and diverse natural products which harbor potent biological activities. Natural products are produced using sets of specialized biosynthetic enzymes encoded by secondary metabolism pathways. Here, we present a two‐step evolutionary model to explain the diversification of biosynthetic pathways that account for the proliferation of these molecules. We argue that the appearance of natural product families has been a slow and infrequent process. The first step led to the original emergence of bioactive molecules and different classes of natural products. However, much of the chemical diversity observed today has resulted from the endless modification of the ancestral biosynthetic pathways. The second step rapidly modulates the pre‐existing biological activities to increase their potency and to adapt to changing environmental conditions. We highlight the importance of enzyme promiscuity in this process, as it facilitates both the incorporation of horizontally transferred genes into secondary metabolic pathways and the functional differentiation of proteins to catalyze novel chemistry. We provide examples where single point mutations or recombination events have been sufficient for new enzymatic activities to emerge. A unique feature in the evolution of microbial secondary metabolism is that gene duplication is not essential but offers opportunities to synthesize more complex metabolites. Microbial natural products are highly important for the pharmaceutical industry due to their unique bioactivities. Therefore, understanding the natural mechanisms leading to the formation of diverse metabolic pathways is vital for future attempts to utilize synthetic biology for the generation of novel molecules.

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Section: Natural Product Biosynthesis and Comparative Genomicsmentioning

confidence: 99%

Section: Direct Modulation Of Enzyme Function In Biosynthetic Gene CLmentioning

confidence: 99%

A pharmaceutical model for the molecular evolution of microbial natural products

Fewer

Metsä‐Ketelä

2019

The FEBS Journal

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“…(3) IPNS (Aspergillus nidulans) is a non-heme Fe(II)dependent oxidase that catalyses the biosynthesis of the precursor of all clinically relevant penicillin and cephalosporin antibiotics, i.e. isopenicillin N (IPN) by four-electron oxidation of its -(l)--aminoadipoyl-(l)-cysteinyl-(d)-valine tripeptide substrate (ACV) (Rabe et al, 2018;Tamanaha et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Anaerobic fixed-target serial crystallography

Rabe

Beale

Butryn

et al. 2020

Int Union Crystallogr J

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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 (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent L-arginine hydroxylase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.

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“…The biosynthesis of isopenicillin N (IPN, 2) from precursor tripeptide δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV, 1; Scheme 1) has been studied in great depth since the discovery of penicillin antibiotics in the early 20th century. [1][2][3] The enzyme responsible for this oxidative bicyclisation reaction is isopenicillin N synthase (IPNS), which catalyses this central step in the biosynthesis and diversification of all penicillin and cephalosporin antibiotics (Scheme 2).…”

Section: Introductionmentioning

confidence: 99%

Isopenicillin N Synthase: Crystallographic Studies

Chapman

Rutledge

2021

ChemBioChem

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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 IPNSÀ FeÀ 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.

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Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in β-lactam biosynthesis

Abstract: The 2OG oxygenases and IPNS contribute to the great structural diversity of β-lactam natural products, employing some remarkable mechanisms.

Cited by 34 publications

References 182 publications

A pharmaceutical model for the molecular evolution of microbial natural products

A pharmaceutical model for the molecular evolution of microbial natural products

Anaerobic fixed-target serial crystallography

Isopenicillin N Synthase: Crystallographic Studies

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