Glutamine‐330 is not essential for activity in isopenicillin N synthase from <i>Aspergillus nidulans</i>

Sami, Malkit; Brown, Toby J. N.; Roach, Peter L.; Schofield, Christopher J.; Baldwin, Jack E.

doi:10.1016/s0014-5793(97)00176-2

Cited by 42 publications

(36 citation statements)

References 15 publications

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“…In contrast, the DAOCS structure reveals only three protein-derived iron ligands and the EXAFS data do not provide evidence for a fourth protein ligand. Thus, the DAOCS studies support mutagenesis studies with IPNS, concluding that iron ligation by a C-terminal residue is not required for catalysis (Sami et al, 1997;Borovok et al, 1996). The structures suggest that the DAOCS C terminus may assist in ordering binding of substrates to the active site.…”

Section: Role Of the C Terminus And Substrate Bindingsupporting

confidence: 53%

“…As noted previously (Sami et al, 1997;Zhang et al, 1997) the errors in apparent K m and k cat values determined from preliminary steady-state kinetic analysis for Fe(II)-dependent oxidizing enzymes may well be signi®cant and caution should be exercised in drawing mechanistic conclusions from relatively small (i.e. less than an order of magnitude) differences.…”

Section: Resultsmentioning

confidence: 95%

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Studies on the active site of deacetoxycephalosporin C synthase

Lloyd¹,

Lee²,

Harlos

et al. 1999

Journal of Molecular Biology

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164

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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.

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Section: Role Of the C Terminus And Substrate Bindingsupporting

confidence: 53%

Section: Resultsmentioning

confidence: 95%

Studies on the active site of deacetoxycephalosporin C synthase

Lloyd¹,

Lee²,

Harlos

et al. 1999

Journal of Molecular Biology

Self Cite

111

164

View full text Add to dashboard Cite

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“…There are precedents for a role of the C-terminus of 2OG oxygenases in substrate binding and selectivity (27)(28)(29)(30). However, removal of the C-terminal residues 403-426 from PHD2 181-426 affects neither its ability to decarboxylate 2OG, nor its ability to hydroxylate CODD or NODD substrates when peptide substrates were assayed separately.…”

Section: Discussionmentioning

confidence: 99%

“…Binding experiments were performed over a range of concentrations and kinetic data were calculated using the BIAevaluation software (GE Healthcare). -Studies on some 2OG oxygenases from the subfamily to which the PHDs belong have revealed a role for the C-terminus of the enzyme in substrate binding and selectivity (23,(27)(28)(29)(30). Alignment of the three human PHD isoform sequences combined with information from the crystal structure of PHD2 181-426 (24) (Fig.…”

Section: Methodsmentioning

confidence: 99%

Kinetic Rationale for Selectivity toward N- and C-terminal Oxygen-dependent Degradation Domain Substrates Mediated by a Loop Region of Hypoxia-Inducible Factor Prolyl Hydroxylases

Flashman

Bagg

Chowdhury

et al. 2008

Journal of Biological Chemistry

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Hydroxylation of two conserved prolyl residues in the N-and C-terminal oxygen-dependent degradation domains (NODD and CODD) of the ␣-subunit of hypoxia-inducible factor (HIF) signals for its degradation via the ubiquitin-proteasome pathway. In human cells, three prolyl hydroxylases (PHDs 1-3) belonging to the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family catalyze prolyl hydroxylation with differing selectivity for CODD and NODD. Sequence analysis of the catalytic domains of the PHDs in the light of crystal structures for PHD2, and results for other 2OG oxygenases, suggested that either the C-terminal region or a loop linking two ␤-strands (␤2 and ␤3 in human PHD2) are important in determining substrate selectivity. Mutation analyses on PHD2 revealed that the ␤2␤3 loop is a major determinant in conferring selectivity for CODD over NODD peptides. A chimeric PHD in which the ␤2␤3 loop of PHD2 was replaced with that of PHD3 displayed an almost complete selectivity for CODD (in competition experiments), as observed for wild-type PHD3. CODD was observed to bind much more tightly to this chimeric protein than the wild type PHD2 catalytic domain.The heterodimeric transcription factor Hypoxia-Inducible Factor (HIF) 2 plays a central role in the response of metazoans to hypoxia (1, 2). Levels of the HIF-␤ subunit are independent of oxygen (3), but under conditions of normoxia, the HIF-␣ subunit is hydroxylated in its oxygen-dependent degradation domain (ODDD), enabling recognition by the von HippelLindau (pVHL) ubiquitin ligase complex and subsequent degradation by the proteasome (4, 5). Under hypoxic conditions, hydroxylation of the HIF-␣ subunit is reduced and it dimerizes with HIF-␤, resulting in the increased expression of hypoxically regulated target genes such as erythropoietin and vascular endothelial growth factor, which enable adaptation to low oxygen concentrations (see reviews in Refs. 6, 7). Hydroxylation of the three HIF-␣ isoforms is catalyzed by prolyl hydroxylases (in humans, PHDs 1-3) (8 -10), belonging to the family of iron(II) and 2-oxoglutarate (2OG)-dependent oxygenases, and occurs at two specific proline residues in the HIF-1␣ ODDD, Pro-402 (N-terminal oxygen-dependent degradation domain, NODD) and Pro-564 (C-terminal oxygen-dependent degradation domain, CODD). HIF-1␣ is also hydroxylated in its C-terminal transactivation domain (C-TAD) at Asn-803, in a reaction catalyzed by another iron and 2OG-dependent oxygenase, Factor Inhibiting HIF (FIH) (11-13). Hydroxylation of Asn-803 prevents binding of HIF to the transcriptional co-activator p300/ CBP in an oxygen-dependent fashion.PHD1 is localized to the nucleus, PHD2 and FIH are commonly found in the cytoplasm, and PHD3 is distributed in both the nucleus and cytoplasm (14). One study on relative activities of recombinant human PHDs, produced by rabbit reticulocyte in vitro transcription and translation, on HIF-1␣, 2␣, and 3␣ has given an order of activity of PHD2 ϭ 3 Ͼ 1 (15) although a different study proposes that PHD2 has the greatest ac...

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“…Deletion of six amino acids from the C terminus of Aspergillus nidulans IPNS (Sami et al, 1997) or Streptomyces clavuligerus DAOCS (Valegard et al, 1998) significantly diminished the activity and it was suggested that the enzymes, which accommodate the active site in a b-sheet barrel, use the C terminus as a protective lid to maintain a hydrophobic environment and to enable proper cofactor binding (Lloyd et al, 1999). In the case of chimeric GA 20-oxidases, a pronounced influence of the C terminus on product selectivity was noticed (Lange et al, 1997).…”

Section: Significance Of the C Terminusmentioning

confidence: 99%

Evolution of Flavone Synthase I from Parsley Flavanone 3β-Hydroxylase by Site-Directed Mutagenesis

et al. 2007

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Flavanone 3b-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/ FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by sitedirected mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the b-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by synelimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction).Flavones and flavonols are the predominant flavonoids found in tissues of Apiaceae species (Harborne, 1971;Harborne and Williams, 1972;Harborne and Baxter, 1999). Significant functions were ascribed to these metabolites for growth and propagation of plants, as well as for adaptation to ecological niches. Flavonoids have been shown to protect from UV radiation, provide pigmentation, mediate the plant's interaction with insects or microbes, and act as feeding deterrents and phytoalexins (Harborne and Williams, 2000;Martens and Mithö fer, 2005). Flavones (i.e. apigenin) are formed by direct 2,3-desaturation of natural flavanones such as (2S)-naringenin (Fig.

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Glutamine‐330 is not essential for activity in isopenicillin N synthase from Aspergillus nidulans

Abstract: © 1997 Federation of European Biochemical Societies.

Cited by 42 publications

References 15 publications

Studies on the active site of deacetoxycephalosporin C synthase

Studies on the active site of deacetoxycephalosporin C synthase

Kinetic Rationale for Selectivity toward N- and C-terminal Oxygen-dependent Degradation Domain Substrates Mediated by a Loop Region of Hypoxia-Inducible Factor Prolyl Hydroxylases

Evolution of Flavone Synthase I from Parsley Flavanone 3β-Hydroxylase by Site-Directed Mutagenesis

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