RESUMO
Teaching old dogs new tricks: Alcohol dehydrogenases (ADHs) may be established redox biocatalysts but they still are good for a few surprises. ADHs can be used to oxidize aldehydes, and this was demonstrated by the oxidative dynamic kinetic resolution of profens. In the presence of a suitable cofactor regeneration system, this reaction can occur with high selectivity.
Assuntos
Álcool Desidrogenase/metabolismo , Aldeídos/metabolismo , Escherichia coli/enzimologia , Álcool Desidrogenase/genética , Álcoois/metabolismo , Escherichia coli/genética , Cinética , Lactobacillus/enzimologia , Oxirredução , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , EstereoisomerismoRESUMO
Quercetinase (QueD) of Streptomyces sp. FLA is an enzyme of the monocupin family and catalyzes the 2,4-dioxygenolytic cleavage of the flavonol quercetin. After expression of the queD gene in Escherichia coli, high specific QueD activity was found in crude cell extracts when the growth medium was supplemented with NiCl 2 or CoCl 2, but not when Mn (2+), Fe (2+), Cu (2+), or Zn (2+) was added. The metal occupancy of Ni- and Co-QueD purified from these cells was
Assuntos
Proteínas de Bactérias/química , Cobalto/química , Dioxigenases/química , Modelos Químicos , Níquel/química , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Catálise , Domínio Catalítico/fisiologia , Dicroísmo Circular/métodos , Dioxigenases/genética , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Streptomyces/genéticaRESUMO
Quercetinase, catalyzing the 2,4-dioxygenolytic cleavage of the flavonol quercetin (3,5,7,3',4'-pentahydroxyflavone) to carbon monoxide and 2-protocatechuoylphloroglucinol carboxylic acid, is encoded by the queD gene in Streptomyces sp. FLA. Because studies on the transcriptional regulation of quercetinase genes are rare, we analyzed the expression of queD in response to quercetin and other carbon compounds. RNA hybridization experiments revealed that transcription of queD is triggered by quercetin and its 3-O-rhamnosylglucoside rutin, but not by the flavonol morin (3,5,7,2',4'-pentahydroxyflavone), the presumed quercetin degradation products protocatechuate and 2,4,6-trihydroxybenzoate or the sugars rhamnose and glucose. Quercetin-induced queD expression was not influenced by the presence of Ni(II), the preferred cofactor of Streptomyces QueD. Reverse transcription-PCR analysis showed a concerted transcription of queD and two putative genes located downstream of queD, which were predicted to code for an amidohydrolase and an esterase. By determination of the transcriptional start site of the queD operon, putative -10 and -35 regions could be identified, suggesting transcription from a sigma70-dependent promoter. Sequence analysis of the queD promoter region indicated possible binding sites for an LmrA/YxaF-like repressor.
Assuntos
Dioxigenases/genética , Dioxigenases/metabolismo , Regulação Bacteriana da Expressão Gênica , Streptomyces/enzimologia , Streptomyces/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Íons/farmacologia , Metais/farmacologia , Dados de Sequência Molecular , Óperon/genética , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Sítio de Iniciação de TranscriçãoRESUMO
The gene queD encoding quercetinase of Streptomyces sp. FLA, a soil isolate related to S. eurythermus (T), was identified. Quercetinases catalyze the 2,4-dioxygenolytic cleavage of 3,5,7,3',4'-pentahydroxyflavone to 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide. The queD gene was expressed in S. lividans and E. coli, and the recombinant hexahistidine-tagged protein (QueDHis(6)) was purified. Several flavonols were converted by QueDHis(6), whereas CO formation from the 2,3-dihydroflavonol taxifolin and the flavone luteolin were not observed. In contrast to bicupin quercetinases from Aspergillus japonicus and Bacillus subtilis, and bicupin pirins showing quercetinase activity, QueD of strain FLA is a monocupin exhibiting 35.9% sequence identity to the C-terminal domain of B. subtilis quercetinase. Its native molecular mass of 63 kDa suggests a multimeric protein. A queD-specific probe hybridized with fragments of genomic DNA of four other quercetin degrading Streptomyces strains, but not with DNA of B. subtilis. Potential ORFs upstream of queD probably code for a serine protease and an endoribonuclease; two ORFs downstream of queD may encode an amidohydrolase and a carboxylesterase. This arrangement suggests that queD is not part of a catabolic gene cluster. Quercetinases might play a major role as detoxifying rather than catabolic enzymes.
Assuntos
Dioxigenases , Flavonóis/metabolismo , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Monóxido de Carbono/metabolismo , Dioxigenases/química , Dioxigenases/genética , Dioxigenases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Dados de Sequência Molecular , Quercetina/metabolismo , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento , Streptomyces lividans/enzimologia , Streptomyces lividans/genéticaRESUMO
Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the mycolic acids-containing actinomycetes, is able to use the lignin degradation products ferulate, vanillate, and protocatechuate as sole carbon sources. The gene cluster responsible for vanillate catabolism was identified and characterized. The vanAB genes encoding vanillate demethylase are organized in an operon together with the vanK gene, coding for a transport system most likely responsible for protocatechuate uptake. While gene disruption mutagenesis revealed that vanillate demethylase is indispensable for ferulate and vanillate utilization, a vanK mutation does not lead to a complete growth arrest but to a decreased growth rate on protocatechuate, indicating that one or more additional protocatechuate transporter(s) are present in C. glutamicum.
