RESUMO
The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have been determined. These structures reveal novel molecular features that provide further insight into the mechanisms behind the sensitivity of this enzyme toward visible light. We propose that a pocket on the si-face of the isoalloxazine ring accommodates oxygen that reacts with photo-excited FAD generating superoxide and a flavin radical that oxidize the isoalloxazine ring C7α methyl group and a nearby tyrosine residue. This tyrosine and key residues surrounding the oxygen pocket are conserved in enzymes from related bacteria, including pathogens such as Staphylococcus aureus. Photo-sensitivity may thus be a widespread feature among bacterial TrxR with the described characteristics, which affords applications in clinical photo-therapy of drug-resistant bacteria.
Assuntos
Lactococcus lactis/enzimologia , Lactococcus lactis/efeitos da radiação , Luz , Estresse Oxidativo , Processos Fotoquímicos , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/química , Flavinas/metabolismo , Redes e Vias Metabólicas , Modelos Moleculares , Conformação Molecular , Oxirredução , Relação Estrutura-AtividadeRESUMO
Natural, nonribosomal cyclotetrapeptides have traditionally been a rich source of inspiration for design of potent histone deacetylase (HDAC) inhibitors. We recently disclosed the total synthesis and full HDAC profiling of the naturally occurring azumamides ( J. Med. Chem. 2013 , 56 , 6512 ). In this work, we investigate the structural requirements for potent HDAC inhibition by macrocyclic peptides using the azumamides along with a series of unnatural analogues obtained through chemical synthesis. By solving solution NMR structures of selected macrocycles and combining these findings with molecular modeling, we pinpoint crucial enzyme-ligand interactions required for potent inhibition of HDAC3. Docking of additional natural products confirmed these features to be generally important. Combined with the structural conservation across HDACs 1-3, this suggests that while cyclotetrapeptides have provided potent and class-selective HDAC inhibitors, it will be challenging to distinguish between the three major class I deacetylases using these chemotypes.
Assuntos
Química Farmacêutica/métodos , Inibidores de Histona Desacetilases/química , Peptídeos Cíclicos/química , Linhagem Celular Tumoral , Simulação por Computador , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores de Histona Desacetilases/síntese química , Humanos , Concentração Inibidora 50 , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Rhamnogalacturonan lyase (RG-lyase) specifically recognizes and cleaves alpha-1,4 glycosidic bonds between L-rhamnose and D-galacturonic acids in the backbone of rhamnogalacturonan-I, a major component of the plant cell wall polysaccharide, pectin. The three-dimensional structure of RG-lyase from Aspergillus aculeatus has been determined to 1.5 A resolution representing the first known structure from polysaccharide lyase family 4 and of an enzyme with this catalytic specificity. The 508-amino acid polypeptide displays a unique arrangement of three distinct modular domains. Each domain shows structural homology to non-catalytic domains from other carbohydrate active enzymes.