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1.
Chem Sci ; 8(2): 1116-1126, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-28451251

RESUMO

A cis-acting enoyl reductase (ER) catalytic domain was isolated from a fungal highly reducing iterative polyketide synthase (HR-iPKS) for the first time and studied in vitro. The ER from the squalestatin tetraketide synthase forms a discrete dimeric protein in solution. The ER shows broad substrate selectivity, reducing enoyl species including both natural and unnatural substrates. Pantetheine-bound substrate thiolesters reacted much faster than the corresponding SNAC thiolesters. The unnatural substrates included Z-olefins, 2-ethyl olefins and pentaketides. Methylation of the substrate modifies the activity of the ER such that the 2,4-dimethyl oct-2-enoyl substrate fits into the active site but cannot be reduced. A new NMR-based assay was developed for the direct observation of the stereochemical preferences at the 4' position of the NADPH cofactor and the C-2 and C-3 positions of the substrates. The assay reveals that the fungal iPKS ER-catalysed reaction is stereochemically identical to that of the vertebrate FAS (vFAS) at the cofactor 4' position and the substrate 3-position, but the high stereoselectivity displayed by intact SQTKS is lost such that reprotonation at the 2-position is unselective by the isolated ER. A 3D model of ER was consistent with these observations and showed that the ER may sequester its final substrate to prevent further chain extension. The results support a developing model for programming by HR-iPKS in which competition for substrates between restrictive and permissive catalytic domains chaperones the growing polyketide to completion, while allowing for errors and evolution.

2.
Chem Commun (Camb) ; 53(10): 1727-1730, 2017 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-28106181

RESUMO

Six potential diketide substrates for the squalestatin tetraketide synthase (SQTKS) dehydratase (DH) domain were synthesised as N-acetyl cysteamine thiolesters (SNAC) and tested in kinetic assays as substrates with an isolated DH domain. 3R-3-hydroxybutyryl SNAC 3R-16 was turned over by the enzyme, but its enantiomer was not. Of the four 2-methyl substrates only 2R,3R-2-methyl-3-hydroxybutyryl SNAC 2R,3R-8 was a substrate. Combined with stereochemical information from the isolated SQTKS enoyl reductase (ER) domain, our results provide a near complete stereochemical description of the first cycle of beta-modification reactions of a fungal highly reducing polyketide synthase (HR-PKS). The results emphasise the close relationship between fungal HR-PKS and vertebrate fatty acid synthases (vFAS).


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Cicloexanonas/química , Dissacarídeos/química , Fungos/enzimologia , Hidroliases/metabolismo , Policetídeo Sintases/metabolismo , Ácidos Tricarboxílicos/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Cicloexanonas/metabolismo , Dissacarídeos/metabolismo , Cinética , Estrutura Molecular , Estereoisomerismo , Ácidos Tricarboxílicos/metabolismo
3.
Invest Ophthalmol Vis Sci ; 54(9): 6052-62, 2013 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-23887803

RESUMO

PURPOSE: Exudative AMD (wet AMD) is treated by monthly injection into the eye of anti-VEGF proteins. VEGF is alternatively spliced to produce numerous isoforms that differ in angiogenic activity. Serine-rich protein kinase-1 (SRPK1) has been identified as a regulator of pro-angiogenic VEGF splicing by phosphorylating serine-rich splicing factor-1 (SRSF1), which binds to VEGF pre-mRNA. We tested the hypothesis that topical (eye drop) SRPK1-selective inhibitors could be generated that reduce pro-angiogenic isoforms, and prevent choroidal neovascularization in vivo. METHODS: Novel inhibitors were tested for SRPK inhibition in vitro, pro-angiogenic VEGF production in RPE cells by PCR and ELISA, and for inhibition of choroidal neovascularisation in mice and rats. RESULTS: A novel disubstituted furan inhibitor was selective for the SRPK family of kinases and reduced expression of pro-angiogenic but not antiangiogenic VEGF isoforms. This inhibitor and previously identified SRPK inhibitors significantly reduced choroidal neovascularisation in vivo. Topical administration of SRPK inhibitors dose-dependently blocked CNV with an EC50 of 9 µM. CONCLUSIONS: These results indicate that novel SRPK1 selective inhibitors could be a potentially novel topical (eye drop) therapeutic for wet AMD.


Assuntos
Neovascularização de Coroide/tratamento farmacológico , Degeneração Macular/complicações , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Epitélio Pigmentado da Retina/patologia , Animais , Células Cultivadas , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Soluções Oftálmicas/administração & dosagem , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/metabolismo , RNA/genética , Splicing de RNA , Ratos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética
4.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 8): 1567-79, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23897479

RESUMO

Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/ß-lactamase-type fold with highest structural similarity to the class A ß-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show ß-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, ß-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ácido Clavulânico/biossíntese , Streptomyces/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Carboxipeptidases/metabolismo , Domínio Catalítico , Cefalosporinas/metabolismo , Ácido Clavulânico/química , Cristalografia por Raios X , Hidrólise , Modelos Moleculares , Penicilinas/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Serina/genética , Streptomyces/genética , beta-Lactamases/química , beta-Lactamases/metabolismo , beta-Lactamas/metabolismo
5.
Org Biomol Chem ; 9(18): 6219-25, 2011 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-21796301

RESUMO

Structural and biochemical analyses reveal how ornithine acetyl-transferases catalyse the reversible transfer of an acetyl-group from a basic (ornithine) to an acidic (glutamate) amino acid by employing a common mechanism involving an acetyl-enzyme intermediate but using different side chain binding modes.


Assuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Mycobacterium tuberculosis/enzimologia , Streptomyces/enzimologia , Sítios de Ligação , Ácido Glutâmico/metabolismo , Modelos Moleculares , Ornitina/metabolismo , Especificidade por Substrato
6.
Chem Commun (Camb) ; 46(29): 5331-3, 2010 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-20552126

RESUMO

The in vivo activity of truncated forms of methylorcinaldehyde synthase shows that the synthase retains a hydrolytic release activity in the absence of reductive chain release and that chain-length is not controlled by the reductive release domain; experiments using a methyltransferase inhibitor suggest that methylation occurs prior to aromatisation.


Assuntos
Biocatálise , Proteínas Fúngicas/química , Policetídeo Sintases/química , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Metilação , Estrutura Molecular , Oxirredução , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo
7.
Clin Neuropsychol ; 7(2): 215-218, 1993 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29022475

RESUMO

The relative difficulty of the logical memory passages on WMS-1, WMS-2, and WMS-R were investigated in different orders of presentation within and between test forms. No evidence of order effects on difficulty level was obtained though, as expected, the second passage on WMS-1 was the most difficult over all orders. The first passages on WMS-1 and WMS-2 and both passages on WMS-R appear to be of equivalent difficulty.

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