Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes

Roach, Peter L.; Clifton, I.J.; Fülöp, Vilmos; Harlos, K.; Barton, Geoffrey J.; Hajdu, János; Andersson, Ingemar; Schofield, Christopher J.; Baldwin, Jack E.

doi:10.1038/375700a0

Cited by 419 publications

(424 citation statements)

References 25 publications

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“…This may be exemplified by spectroscopic studies when NO directly binds to the ferrous ion in protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase (Arciero et al, 1985) or to isopenicillin N synthase (Roach et al, 1995). These enzymes coordinate Fe 2ϩ in their catalytic domain in a 2-histidine-1-carboxylate facial triad that is the defining structural motif of mononuclear nonheme iron(II) enzymes (Hegg and Que, 1997).…”

Section: Discussionmentioning

confidence: 99%

Nitric Oxide Impairs Normoxic Degradation of HIF-1α by Inhibition of Prolyl Hydroxylases

Metzen

Zhou

Jelkmann

et al. 2003

MBoC

379

261

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Hypoxia inducible factor-1 (HIF-1) is the master regulator of metabolic adaptation to hypoxia. It is appreciated that HIF-1␣ accumulation is achieved under normoxic conditions by e.g., nitric oxide. We determined molecular mechanisms of HIF-1␣ accumulation under the impact of S-nitrosoglutathione (GSNO). In human embryonic kidney cells GSNO provoked nuclear accumulation of HIF-1␣. This appeared unrelated to gene transcription and protein translation, thus pointing to inhibition of HIF-1␣ degradation. Indeed, GSNO as well as the hypoxia mimic CoCl 2 decreased ubiquitination of HIF-1␣ and GSNO-induced HIF-1␣ failed to coimmunoprecipitate with pVHL (von Hippel Lindau protein). Considering that HIF-1␣-pVHL interactions require prolyl hydroxylation of HIF-1␣, we went on to demonstrate inhibition of HIF-1␣ prolyl hydroxylases (PHDs) by GSNO. In vitro HIF-1␣-pVHL interactions revealed that GSNO dose-dependently inhibits PHD activity but not the interaction of a synthetic peptide resembling the hydroxylated oxygen-dependent degradation domain of HIF-1␣ with pVHL. We conclude that GSNO-attenuated prolyl hydroxylase activity accounts for HIF-1␣ accumulation under conditions of NO formation during normoxia and that PHD activity is subject to regulation by NO. INTRODUCTIONThe heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1) plays a central role in adaptation to decreased oxygen availability (Semenza, 2002;Wenger, 2002;Brü ne and Zhou, 2003). HIF-1 is composed of the two basic helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) proteins HIF-1␣ and the aryl hydrocarbon receptor nuclear translocator (ARNT), also known as HIF-1␤ (Wang and Semenza, 1995). In many cell types, the mRNA of both HIF-1␣ and HIF-1␤ appear permanently expressed and HIF-1␤ protein is constitutively present. However, HIF-1␣ protein is kept at a low or undetectable level under normoxia. In hypoxia, HIF-1␣ is strikingly induced, translocates to the nucleus, and dimerizes with HIF-1␤ to form HIF-1, which binds to hypoxiaresponsive elements (HRE) in regulatory regions of an impressive array of target genes involved in angiogenesis, erythropoiesis, vasomotor control, and energy metabolism as well as in cell survival decisions (for references, see Maxwell et al., 2001;Wenger, 2002; Zhu et al., 2002). This makes HIF-1 the master regulator of oxygen homeostasis to meet cell and tissue requirements in a situation of oxygen deficiency.In normoxia HIF-1␣ is bound to the von Hippel Lindau protein (pVHL) (Maxwell et al., 1999;Hon et al., 2002;Min et al., 2002), which is the substrate recognizing component of an E3 ubiquitin ligase complex (Cockman et al., 2000;Ohh et al., 2000;Tanimoto et al., 2000). Consequently, HIF-1␣ is polyubiquitinated and degraded by the 26S proteasome system, thus accounting for its low normoxic level of expression (Salceda and Caro, 1997;Huang et al., 1998;Kallio et al., 1999). The requirement of pVHL for HIF-1␣ degradation is underscored in cells that do not contain a functional pVHL, which leads to high levels of HIF-1␣ in normox...

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Section: Discussionmentioning

confidence: 99%

Nitric Oxide Impairs Normoxic Degradation of HIF-1α by Inhibition of Prolyl Hydroxylases

Metzen

Zhou

Jelkmann

et al. 2003

MBoC

379

261

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“…The side‐chain amide carbonyl oxygen of Gln330, the penultimate residue of the enzyme, ligates to the active site metal only in the absence of substrate, as observed in the structure of the IPNS:Mn II complex (Figure S1 a) 5. In the structure of the anaerobic IPNS:Fe II :ACV complex6 and structures with substrate analogues,13, 26, 27, 28, 29 the side‐chain of Gln330 is displaced from the metal to enable ligation of the substrate sulfur.…”

Section: Introductionmentioning

confidence: 87%

“…The mechanism of IPNS has been studied using a wide range of ACV analogues in solution,12 in crystallisation studies,5, 6, 13, 14 and by turnover within the crystalline protein (with reaction initiated by exposing anaerobic crystals to pressurised oxygen gas) 7, 15, 16, 17, 18. IPNS catalyses an array of different oxidation reactions both in solution and in crystals; many of the ACV analogues studied, particularly those altered in the third residue (valine), are oxidised to alternative cyclic and acyclic products.…”

Section: Introductionmentioning

confidence: 99%

Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non‐Heme Iron Oxidases

McNeill

Brown

Sami

et al. 2017

Chemistry A European J

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Isopenicillin N synthase (IPNS) catalyses the four‐electron oxidation of a tripeptide, l‐δ‐(α‐aminoadipoyl)‐l‐cysteinyl‐d‐valine (ACV), to give isopenicillin N (IPN), the first‐formed β‐lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co‐substrate. In the absence of substrate, the carbonyl oxygen of the side‐chain amide of the penultimate residue, Gln330, co‐ordinates to the active‐site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C‐terminal residues, which move to take up a conformation that extends the final α‐helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C‐terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side‐chains around the ACV binding pocket is important in catalysis. Deletion of seven C‐terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC‐MS and NMR analyses to be the ene‐thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl β‐carbon of the other. A mechanism for its formation is proposed, supported by an X‐ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl β‐carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non‐heme iron oxidases in general.

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“…Like PDO, the reaction mechanism of isopencillin N-synthase involves Fe-S-peptide interactions as the linear cysteinyl-tripeptide substrate is transformed into isopenicillin N via desaturative ring closure and concomitant reduction of O 2 to water (22). Fe-SCys interactions are also important in the cysteine dioxygenase-catalyzed dioxygenation of cysteine to cysteinesulfinic acid (23).…”

Section: Discussionmentioning

confidence: 99%

Mechanism-based inhibition of human persulfide dioxygenase by γ-glutamyl-homocysteinyl-glycine

Kabil

Motl

Strack

et al. 2018

Journal of Biological Chemistry

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Hydrogen sulfide (H 2 S) is a signaling molecule with many beneficial effects. However, its cellular concentration is strictly regulated to avoid toxicity. Persulfide dioxygenase (PDO or ETHE1) is a mononuclear non-heme ironcontaining protein in the sulfide oxidation pathway catalyzing the conversion of glutathione persulfide (GSSH) to sulfite and glutathione. PDO mutations result in the autosomal recessive disorder, ethylmalonic encephalopathy (EE). Here, we developed γ-glutamyl-homocysteinyl-glycine (GHcySH) in which the cysteinyl moiety in glutathione is substituted with homocysteine, as a mechanismbased PDO inhibitor. Human PDO used GHcySH as an alternate substrate and converted it to GHcy-SO 2 H, mimicking GS-SO 2 H, the putative oxygenated intermediate formed with the natural substrate. Since GHcy-SO 2 H contains a C-S bond rather than an S-S bond in GS-SO 2 H, it failed to undergo the final hydrolysis step in the catalytic cycle, leading to PDO inhibition. We also characterized the biochemical penalties incurred by the L55P, T136A, C161Y and R163W mutations reported in EE patients. The variants displayed lower iron content (1.4-to 11-fold) and lower thermal stability (1.2-to 1.7-fold) than wild-type PDO. They also exhibited varying degrees of catalytic impairment; the k cat /K m values for R163W, L55P and C161Y PDOs were 18-, 42-and 65-fold lower, respectively and the T136A variant was most affected with a 200-fold lower k cat /K m . Like wild-type enzyme, these variants were inhibited by GHcySH. This study provides the first characterization of an intermediate in the PDO-catalyzed reaction and reports on deficits associated with EE-linked mutations that are distal from the active site.Ethylmalonic encephalopathy (EE) 1 is an autosomal recessive disorder that is associated with pathological effects in the brain, gastrointestinal tract and peripheral vessels (1-3). It results in acrocyanosis, petechiae, hemorrhagic diarrhea, developmental delay and progressive neurological failure leading to necrotic lesions in the deep gray matter of the brain. Patients with EE usually succumb to the disease within the first decade of life (4). The clinical profile of EE includes elevated ethylmalonic acid in urine, C4 and C5 acrylcarnitines in blood and a deficiency of cytochrome c oxidase in brain and muscle (5). EE is caused by mutations in the ethe1 gene that encodes persulfide dioxygenase (PDO or ETHE1). Over 20 mutations have been described in the ethe1 gene, of which a subset represents missense mutations (3,4,6).PDO is a mitochondrial matrix protein that participates in the mitochondrial sulfide oxidation pathway, which converts H 2 S to the end products, thiosulfate and sulfate (7,8). In addition to PDO, the other enzymes involved in the mitochondrial sulfur oxidation pathway are sulfide quinone oxidoreductase (9), rhodanese (10), and sulfite oxidase (11). PDO catalyzes the second step in the pathway, i.e. the oxidation of glutathione persulfide (GSSH) to sulfite (Eq. 1) (12). Sulfite is either oxidized b...

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Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes

Cited by 419 publications

References 25 publications

Nitric Oxide Impairs Normoxic Degradation of HIF-1α by Inhibition of Prolyl Hydroxylases

Nitric Oxide Impairs Normoxic Degradation of HIF-1α by Inhibition of Prolyl Hydroxylases

Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non‐Heme Iron Oxidases

Mechanism-based inhibition of human persulfide dioxygenase by γ-glutamyl-homocysteinyl-glycine

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