RESUMEN
The pcd gene from Flavobacterium lutescens IFO3084 encoding Delta'-piperideine-6-carboxylate dehydrogenase (PCD) was cloned, sequenced, and expressed in Escherichia coli. The deduced amino acid sequence of PCD from F. lutescens IFO3084 showed strong similarity to that from Streptomyces clavuligerus. The molecular mass of the recombinant PCD was estimated to be approximately 58,000 Da by SDS-PAGE and native PAGE, which indicated that the enzyme molecule is a monomer. The in vitro analysis of L-alpha-aminoadipic acid (L-AAA) production showed that L-AAA is synthesized from L-lysine in two steps catalyzed by L-lysine 6-aminotransferase (LAT) and PCD from F. lutescens IFO3084.
Asunto(s)
Proteínas Bacterianas , Flavobacterium/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Datos de Secuencia Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
L-Lysine 6-aminotransferase (LAT) is an enzyme involved in L-lysine catabolism in a wide range of living organisms. LAT from Flavobacterium lutescens IFO3084 was purified, and its structural gene (lat) was cloned, sequenced and expressed in Escherichia coli. Native PAGE analysis of purified LAT gave a single band corresponding to a molecular weight of about 110,000. lat encoded a protein of 493 amino acids with a deduced molecular weight of 53,200, which is very close to that of purified LAT determined on SDS-PAGE. Expression of lat in E. coli revealed that lat encodes a single subunit protein leading to LAT activity. These data suggested that LAT from F. lutescens IFO3084, like most other aminotransferases, is derived from a single ORF and is active as a homodimer.
Asunto(s)
Flavobacterium/enzimología , Genes/genética , Transaminasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/química , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Escherichia coli/enzimología , Expresión Génica , Vectores Genéticos , L-Lisina 6-Transaminasa , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transaminasas/aislamiento & purificaciónRESUMEN
The structures of rhodopeptins, novel antifungal peptides, were determined on the basis of physico-chemical analyses of the intact molecules and their acid hydrolysates. The structures of rhodopeptins C1, C2, C3, C4 and B5 were determined to be cyclo (-Gly-L-Orn-L-Val-3-amino-10-methyldodecanoyl-), cyclo (-Gly-L-Orn-L-Ile-3-amino-10-methyldodecanoyl-), cyclo (-Gly-L-Orn-L-Val-3-amino-12-methyltridecanoyl-), cyclo (-Gly-L-Orn-L-Val-3-amino- 12-methyltetradecanoyl-) and cyclo (-Gly-L-Lys-L-Val-3-amino-13-methyltetradecanoyl-), respectively. They are novel cyclic tetrapeptides containing a lipophilic beta-amino acid.
Asunto(s)
Antifúngicos/química , Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificación , Péptidos Cíclicos/farmacología , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Rhodococcus/metabolismo , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
Total syntheses of cyclo (-Gly-L-Lys-L-Val-(R)-3-aminododecanoyl-); LV9nA and its diastereomer cyclo (-Gly-L-Lys-L-Val-(S)-3-aminododecanoyl-); LV9nB, congeners of rhodopeptin B5 on beta-amino acid moiety, were achieved. The beta-amino acid moiety was prepared as a racemate by the thermal Michael addition of an amine to alpha,beta-unsaturated ester. The racemic beta-amino acids were converted to their L-Valylamide derivatives and the obtained diastereomers were separated. Coupling of both diastereomers, L-Val-beta-amino acids with Gly-L-Lys gave linear tetrapeptides, and tetrapeptides were cyclized by diphenylphosphoryl azide (DPPA) method between C-terminus of beta-amino acid and N-terminus of Gly to give cyclic tetrapeptides. The deprotected cyclic tetrapeptides, LV9nA and LV9nB, both exhibited almost the same antifungal activity as the naturally obtained rhodopeptins. Furthermore, comparison of the 1H NMR spectra of two congeners and rhodopeptin B5 suggested that the stereochemistry of beta-amino acid moiety in natural rhodopeptin B5 has (R)-configuration.
Asunto(s)
Antifúngicos/síntesis química , Antifúngicos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Antifúngicos/química , Bioquímica/métodos , Evaluación Preclínica de Medicamentos , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Rhodococcus/metabolismo , Relación Estructura-ActividadRESUMEN
Mer-WF3010, a new member of the papulacandin family, was isolated from the mycelia of Phialophora cyclaminis Mer-WF3010 (FERM P-11475). The molecular formula of Mer-WF3010 was determined as C45H60O16.
Asunto(s)
Aminoglicósidos , Antibacterianos/aislamiento & purificación , Antifúngicos/aislamiento & purificación , Phialophora/metabolismo , Antibacterianos/biosíntesis , Antibacterianos/farmacología , Antifúngicos/biosíntesis , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Equinocandinas , FermentaciónRESUMEN
Five novel cyclic tetrapeptides, named rhodopeptin C1, C2, C3, C4 and B5, were isolated from a strain named Rhodococcus sp. Mer-N1033. They are a novel type of cyclic tetrapeptide composed of a beta-amino acid and three usual alpha-amino acids. Rhodopeptins show high in vitro antifungal activity against Candida albicans and Cryptococcus neoformans, whereas they show no activity against bacteria.
Asunto(s)
Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificación , Péptidos Cíclicos/farmacología , Rhodococcus/clasificación , Rhodococcus/metabolismo , Animales , Antifúngicos/química , Candida albicans/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Fermentación , Leucemia L1210/tratamiento farmacológico , Pruebas de Sensibilidad MicrobianaRESUMEN
Penicillin X methyl ester was transformed into three types of dimer by laccase from Coriolus versicolor. The dimers are considered to be formed by free-radical addition of phenoxy radicals produced by laccase. The enzyme reaction with the ester as substrate was more suitable for forming dimers than that with the sodium salt as substrate. Penicillin X pivaloyloxymethyl ester was also transformed into a dimer, which had antibacterial activity in the presence of esterase.