RESUMEN
A serine residue Ser463, required for proper function of E. coli -glutamyltranspeptidase (EcGGT) was identified by site-directed mutagenesis on the basis of sequence alignment of human, pig, rat, and three bacterial enzymes. Thr-, Asp-, and Lys-substituted variants were overexpressed in E. coli M15 cells and the recombinant proteins were purified to near homogeneity by nickel-chelate chromatography. With the exception of S463T, the other two variants completely lost GGT activity, implying the importance of this residue in EcGGT. Moreover, substitution of Ser463 with either Lys or Asp impaired the capability of autocatalytic processing of the precursor into - and -subunit. Computer modeling showed that the critical bonding distance of Gln390 C-Thr391 OG1 was significantly increased in S463D and S463K, indicating that these distance changes might be responsible for the lack of enzyme maturation. Measurements of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues in S463D and S463K, while circular dichroism (CD) spectra were nearly identical for wild-type and all mutant enzymes. The temperature-dependent signal in the far-UV region for S463T was consistent with that of wild-type enzyme, but S463D and S463K showed a different sensitivity towards temperature-induced denaturation. These results implied that a significant conformational change occurred as a result of Asp- and Lys-substitution.
Asunto(s)
Secuencia de Aminoácidos , Ácido Aspártico/química , Catálisis , Dicroismo Circular , Escherichia coli/enzimología , Glutamina/química , Lisina/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Serina/química , Espectrometría de Fluorescencia/métodos , Treonina/química , Triptófano/química , gamma-Glutamiltransferasa/química , gamma-Glutamiltransferasa/genéticaRESUMEN
Sequence comparison showed that residues Thr407, Asp433, and Met464 in the small subunit of Escherichia coli gamma-glutamyltranspeptidase (EcGGT) were conserved in the aligned enzymes. In this study, we further investigated the functional significance of these conserved residues by site-directed mutagenesis. The wild-type and mutant enzymes were overexpressed in the recombinant E. coli M15 cells and purified to near homogeneity by Ni2+-NTA resin. Except M464L, other mutants had shown no GGT activity under enzyme assay conditions and activity staining. Furthermore, mutations on these residues impaired the capability of autocatalytic processing of the enzyme. Based on these observations, it is concluded that these residues play an important role in the enzyme maturation.
Asunto(s)
Secuencia de Aminoácidos , Aminoácidos/genética , Secuencia Conservada , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/métodos , Mutación , gamma-Glutamiltransferasa/genéticaRESUMEN
A truncated Escherichia coli Novablue gamma-glutamyltranspeptidase (EcGGT) gene, lacking the first 48-bp coding sequence for part of the signal sequence, was amplified by polymerase chain reaction (PCR) and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His6-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 degrees C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 21 kDa respectively by SDS-PAGE, indicating the precursor EcGGT still undergoes the post-translational processing even in the truncation of signal sequence. His6-tagged EcGGT migrated relative to the molecular mass of approximately 120 kDa and its heterodimeric structure was confirmed by a native-PAGE gel.