RESUMO
Proper subcellular localization of focal adhesion kinase (FAK) is crucial for many cellular processes. It remains, however, unclear how FAK activity is regulated at subcellular compartments. To visualize the FAK activity at different membrane microdomains, we develop a fluorescence resonance energy transfer (FRET)-based FAK biosensor, and target it into or outside of detergent-resistant membrane (DRM) regions at the plasma membrane. Here we show that, on cell adhesion to extracellular matrix proteins or stimulation by platelet-derived growth factor (PDGF), the FRET responses of DRM-targeting FAK biosensor are stronger than that at non-DRM regions, suggesting that FAK activation can occur at DRM microdomains. Further experiments reveal that the PDGF-induced FAK activation is mediated and maintained by Src activity, whereas FAK activation on cell adhesion is independent of, and in fact essential for the Src activation. Therefore, FAK is activated at membrane microdomains with distinct activation mechanisms in response to different physiological stimuli.
Assuntos
Membrana Celular/química , Membrana Celular/enzimologia , Transferência Ressonante de Energia de Fluorescência/métodos , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Animais , Adesão Celular , Membrana Celular/genética , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/genética , Camundongos , Camundongos Knockout , Fator de Crescimento Derivado de Plaquetas/metabolismo , Estrutura Terciária de ProteínaRESUMO
The chemoenzymatic installation of the clinically valuable (S)-4-amino-2-hydroxybutyryl side chain onto a number of 2-deoxystreptamine-containing aminoglycosides is described using the purified Bacillus circulans biosynthetic enzymes BtrH and BtrG in combination with a synthetic acyl-SNAC surrogate substrate.
Assuntos
Aminoglicosídeos/síntese química , Antibacterianos/síntese química , Acilação , Proteínas de Bactérias/metabolismo , Sulfato de Butirosina/síntese química , Sulfato de Butirosina/farmacologia , Indicadores e Reagentes , Proteínas de Membrana Transportadoras/metabolismo , Transaminases/metabolismoAssuntos
Antibacterianos/biossíntese , Bactérias/enzimologia , Transglutaminases/metabolismo , Bactérias/genética , Bactérias/metabolismo , Catálise , Genes Bacterianos/genética , Família Multigênica/genética , Peptídeo Sintases/química , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Polienos/química , Polienos/metabolismo , Pirróis/química , Pirróis/metabolismo , Transglutaminases/química , Transglutaminases/genéticaRESUMO
Butirosin, an aminoglycoside antibiotic produced by Bacillus circulans, bears the unique (S)-4-amino-2-hydroxybutyrate (AHBA) side chain, which protects the antibiotic from several common resistance mechanisms. The AHBA side chain is advantageously incorporated into clinically valuable antibiotics such as amikacin and arbekacin by synthetic methods. Therefore, it is of significant interest to explore the biosynthetic origins of this useful moiety. We report here that the AHBA side chain of butirosin is transferred from the acyl carrier protein (ACP) BtrI to the parent aminoglycoside ribostamycin as a gamma-glutamylated dipeptide by the ACP:aminoglycoside acyltransferase BtrH. The protective gamma-glutamyl group is then cleaved by BtrG via an uncommon gamma-glutamyl cyclotransferase mechanism. The application of this pathway to the in vitro enzymatic production of novel AHBA-bearing aminoglycosides is explored with encouraging implications for the preparation of unnatural antibiotics via directed biosynthesis.
Assuntos
Bacillus/metabolismo , Sulfato de Butirosina/biossíntese , Proteína de Transporte de Acila/metabolismo , Aminoácidos/metabolismo , Aminoglicosídeos/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sulfato de Butirosina/química , Sulfato de Butirosina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The proteins Neo-11 and Neo-18 encoded in the neomycin gene cluster (neo) of Streptomyces fradiae NCIMB 8233 have been characterized as glucosaminyl-6'-oxidase and 6'-oxoglucosaminyl:L-glutamate aminotransferase, respectively. The joint activity of Neo-11 and Neo-18 is responsible for the conversion of paromamine to neamine in the biosynthetic pathway of neomycin through a mechanism of FAD-dependent dehydrogenation followed by a pyridoxal-5'-phosphate-mediated transamination. Neo-18 is also shown to catalyze deamination at C-6''' of neomycin, thus suggesting bifunctional roles of the two enzymes in the formation of both neosamine rings of neomycin. The product of the btrB gene, a homologue of neo-18 in the butirosin biosynthetic gene cluster (btr) in Bacillus circulans, exhibits the same activity as Neo-18; this indicates that there is a similar reaction sequence in both butirosin and neomycin biosynthesis.
Assuntos
Antibacterianos/biossíntese , Sulfato de Butirosina/biossíntese , Glucosamina/análogos & derivados , Neomicina/biossíntese , Oxirredutases/química , Transaminases/química , Bacillus/enzimologia , Bacillus/genética , Sequência de Carboidratos , Ciclização , Glucosamina/química , Glucosamina/classificação , Dados de Sequência Molecular , Família Multigênica , Oxirredutases/genética , Oxirredutases/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Transaminases/genética , Transaminases/metabolismoAssuntos
Aminoglicosídeos/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Sulfato de Butirosina/biossíntese , Glucosamina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sulfato de Butirosina/química , Histona Desacetilases/química , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The 2-deoxystreptamine-containing aminoglycosides are an important class of clinically valuable antibiotics. A deep understanding of the biosynthesis of these natural products is required to enable efforts to rationally manipulate and engineer the biological production of novel aminoglycosides. This review discusses the development of our biosynthetic knowledge over the past half-century, with emphasis on the relatively recent contributions of molecular biology to the elucidation of these biosynthetic pathways.
Assuntos
Aminoglicosídeos/biossíntese , Antibacterianos/biossíntese , Aminoglicosídeos/química , Antibacterianos/química , Hexosaminas/biossíntese , Estrutura MolecularRESUMO
Butirosins A and B are naturally occurring aminoglycoside antibiotics that have a (2S)-4-amino-2-hydroxybutyrate (AHBA) side chain. Semisynthetic addition of AHBA to clinically valuable aminoglycoside antibiotics has been shown both to improve their pharmacological properties and to prevent their deactivation by a number of aminoglycoside-modifying enzymes involved in bacterial resistance. We report here that the biosynthesis of AHBA from L-glutamate, encoded within a previously identified butirosin biosynthetic gene cluster, proceeds via intermediates tethered to a specific acyl carrier protein (ACP). Five components of the pathway have been purified and characterized, including the ACP (BtrI), an ATP-dependent ligase (BtrJ), a pyridoxal phosphate-dependent decarboxylase (BtrK), and a two-component flavin-dependent monooxygenase system (BtrO and the previously unreported BtrV). The proposed biosynthetic pathway includes a gamma-glutamylation of an ACP-derived gamma-aminobutyrate intermediate, possibly a rare example of protective group chemistry in biosynthesis.