RESUMO
The gene product of orf50 from actinophage µ1/6 of Streptomyces aureofaciens is a putative endolysin, Lyt µ1/6. It has a two-domain modular structure, consisting of an N-terminal catalytic and a C-terminal cell wall binding domain (CBD). Comparative analysis of Streptomyces phage endolysins revealed that they all have a modular structure and contain functional C-terminal domains with conserved amino acids, probably associated with their binding function. A blast analysis of Lyt µ1/6 in conjunction with secondary and tertiary structure prediction disclosed the presence of a PG_binding_1 domain within the CBD. The sequence of the C-terminal domain of lyt µ1/6 and truncated forms of it were cloned and expressed in Escherichia coli. The ability of these CBD variants fused to GFP to bind to the surface of S. aureofaciens NMU was shown by specific binding assays.
Assuntos
Bacteriófagos/genética , Endopeptidases/química , Streptomyces aureofaciens/virologia , Sequência de Aminoácidos , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Streptomyces aureofaciens/metabolismoRESUMO
Glycoside hydrolase family 77 (GH77) contains prokaryotic amylomaltases and plant-disproportionating enzymes (both possessing the 4-alpha-glucanotransferase activity; EC 2.4.1.25). Together with GH13 and GH70, it forms the clan GH-H, known as the alpha-amylase family. Bioinformatics analysis revealed that the putative GH77 amylomaltase (MalQ) from the Lyme disease spirochaete Borrelia burgdorferi genome (BB0166) contains several amino acid substitutions in the positions that are important and conserved in all GH77 amylomaltases. The most important mutation concerned the functionally important arginine positioned two residues before the catalytic nucleophile that is replaced by lysine in B. burgdorferi MalQ. Similar remarkable substitutions were found in two other putative GH77 amylomaltases from related borreliae. In order to confirm the exclusive sequence features and to verify the eventual enzymatic activity, the malQ gene from B. burgdorferi was amplified using PCR. A c. 1.5-kb amplified DNA fragment was sequenced, cloned and expressed in Escherichia coli, and the resulting recombinant protein was preliminarily characterized for its activity towards glucose (G1) and a series of malto-oligosaccharides (G2-G7). This study confirmed that the remarkable substitution of the arginine really exists and the GH77 MalQ protein from B. burgdorferi is a functional amylomaltase because it is able to hydrolyse the malto-oligosaccharides as well as to form their longer transglycosylation products.
