Effects of C-terminal truncation on autocatalytic processing of Bacillus licheniformis γ-glutamyl transpeptidase

Abstract

The role of the C-terminal region of Bacillus licheniformis gamma-glutamyl transpeptidase (BlGGT) was investigated by deletion analysis. Seven C-terminally truncated BlGGTs lacking 581-585, 577-585, 576-585, 566-585, 558-585, 523-585, and 479-585 amino acids, respectively, were generated by site-directed mutagenesis. Deletion of the last nine amino acids had no appreciable effect on the autocatalytic processing of the enzyme, and the engineered protein was active towards the synthetic substrate L-gamma-glutamyl-p-nitroanilide. However, a further deletion to Val576 impaired the autocatalytic processing. In vitro maturation experiments showed that the truncated BlGGT precursors, pro-Delta(576-585), pro-Delta(566-585), and pro-Delta(558-585), could partially precede a time-dependent autocatalytic process to generate the L- and S-subunits, and these proteins showed a dramatic decrease in catalytic activity with respect to the wild-type enzyme. The parental enzyme (BlGGT-4aa) and BlGGT were unfolded biphasically by guanidine hydrochloride (GdnCl), but Delta(577-585), Delta(576-585), Delta(566-585), Delta(558-585), Delta(523-585), and Delta(479-585) followed a monophasic unfolding process and showed a sequential reduction in the GdnCl concentration corresponding to half effect and DeltaG(0) for the unfolding. BlGGT-4aa and BlGGT sedimented at ~4.85 S and had a heterodimeric structure of approximately 65.23 kDa in solution, and this structure was conserved in all of the truncated proteins. The frictional ratio (f/f(o)) of BlGGT-4aa, BlGGT, Delta(581-585), and Delta(577-585) was 1.58, 1.57, 1.46, and 1.39, respectively, whereas the remaining enzymes existed exclusively as precursor form with a ratio of less than 1.18. Taken together, these results provide direct evidence for the functional role of the C-terminal region in the autocatalytic processing of BlGGT.

Specific biochemical amniotic fluid pattern of fetal isolated esophageal atresia

“…AMPs comprise a large family of biologically important zinc enzymes playing different roles in the regulation of blood pressure, antigen presentation, memory, cell cycle, pregnancy, and angiogenesis. Numerous polymorphisms are present within the human species, and mutations can result in hypertension or preeclampsia (22,23). Literature data are scarce concerning the biochemical properties of AMP, notably its biochemical stability is not reported and reports mostly focused on its multiple polymorphisms and their consequences for the phenotype.…”

Covalent Immobilization of Bacillus licheniformis γ-Glutamyl Transpeptidase on Aldehyde-Functionalized Magnetic Nanoparticles

“…Deletion analyses of the recombinant enzyme have demonstrated that both N- and C-terminal sequences are crucial for the expression of its active form in host cells [33,34]. Given that nano-sized magnetic particles have recently received considerable attention for chemical cross-linking of enzymes [22,34–38], the use of surface-modified magnetic nanoparticles to covalently immobilize Bl GGT should be a persuasive approach for obtaining stable and reusable biocatalyst preparations. In this work, we coated the prepared Fe 3 O 4 nanoparticles with aminosilane and, subsequently, immobilized Bl GGT on the nanomaterial through a glutaraldehyde coupling reaction (Figure 1).…”

Heterogeneous nucleation helps the search for initial crystallization conditions of γ-glutamyl transpeptidase fromBacillus licheniformis

“…It is a heterodimer that efficiently cleaves l--glutamyl-p-nitroanilide (l--Glu-p-NA) with a k cat of 105 s À1 and a K m of 21 mM. BlGT is a moderately thermostable enzyme and is among the most stable characterized -GTs (Pica et al, 2012(Pica et al, , 2013, showing 80% of its activity at 328 K and a denaturation temperature of 333-338 K (Chang et al, 2010;Yang et al, 2011). Deletion analyses of the recombinant enzyme have demonstrated that both the Nterminal and the C-terminal sequences are crucial for the expression of its active form in host cells and for its thermal stability (Lin et al, 2008;Chang et al, 2010).…”