Biophysical characterization of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases: A comparative analysis

Yang, Jia-Ci; Liang, Wan-Chi; Chen, Yiyu; Chi, Meng-Chun; Lo, Huei-Fen; Chen, Hsiang-Ling; Lin, Long-Liu

doi:10.1016/j.ijbiomac.2011.01.006

Cited by 23 publications

(15 citation statements)

References 35 publications

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“…GGTP is found in organisms from bacteria to mammals and plants. Literature data concerning GGTP biochemical properties showed evidence of high thermostability, independence of pH variations, and stability in high-salt conditions (16)(17)(18)(19)(20)(21). 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.…”

Section: Discussionmentioning

confidence: 99%

“…Results are expressed in MoM, corresponding to the ratio between the observed raw value and the median raw value defined for the same gestational age. For the study of GGTP and AMP half-lives in AF, we used four pools of control AF samples at different GGTP levels (200, 500, 700, and 1000 IU/l) and AMP levels (20,60,80, and 100 IU/l) incubated at 37 °C. An aliquot was tested at 7-to 10-day intervals for AMP and GGTP activities up to 61 d. Finally, based on previous data (14) and on the GGTP and AMP half-lives we observed herein, we have constructed a theoretical model explaining AF-GGTP and AMP kinetics as a function of gestational age in controls and in EA cases.…”

Section: Af Biochemistrymentioning

confidence: 99%

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Specific biochemical amniotic fluid pattern of fetal isolated esophageal atresia

et al. 2013

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Section: Discussionmentioning

confidence: 99%

Section: Af Biochemistrymentioning

confidence: 99%

Specific biochemical amniotic fluid pattern of fetal isolated esophageal atresia

et al. 2013

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“…The irreversible inactivation of Bl GGT by heat has already been reported to follow the one-state process [49]. As shown in Figure 5C, the wild-type enzyme started to denature at around 48.1 °C and was transformed from its native state into a completely unfolded polypeptide chain at 70.2 °C.…”

Section: Resultsmentioning

confidence: 69%

Mutational Analysis of a Highly Conserved PLSSMXP Sequence in the Small Subunit of Bacillus licheniformis γ-Glutamyltranspeptidase

Chi

Lin

et al. 2019

Biomolecules

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A highly conserved 458PLSSMXP464 sequence in the small subunit (S-subunit) of an industrially important Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was identified by sequence alignment. Molecular structures of the precursor mimic and the mature form of BlGGT clearly reveal that this peptide sequence is in close spatial proximity to the self-processing and catalytic sites of the enzyme. To probe the role of this conserved sequence, ten mutant enzymes of BlGGT were created through a series of deletion and alanine-scanning mutagenesis. SDS-PAGE and densitometric analyses showed that the intrinsic ability of BlGGT to undergo autocatalytic processing was detrimentally affected by the deletion-associated mutations. However, loss of self-activating capacity was not obviously observed in most of the Ala-replacement mutants. The Ala-replacement mutants had a specific activity comparable to or greater than that of the wild-type enzyme; conversely, all deletion mutants completely lost their enzymatic activity. As compared with BlGGT, S460A and S461S showed greatly enhanced kcat/Km values by 2.73- and 2.67-fold, respectively. The intrinsic tryptophan fluorescence and circular dichroism spectral profiles of Ala-replacement and deletion mutants were typically similar to those of BlGGT. However, heat and guanidine hydrochloride-induced unfolding transitions of the deletion-associated mutant proteins were severely reduced as compared with the wild-type enzyme. The predictive mutant models suggest that the microenvironments required for both self-activation and catalytic reaction of BlGGT can be altered upon mutations.

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“…Wild‐type and mutant enzymes were over‐expressed in recombinant E. coli M15 cells and purified as described previously [45,46]. Protein purity and autocatalytic processing were checked by SDS–PAGE with the Laemmli buffer system [47].…”

Section: Methodsmentioning

confidence: 99%

Experimental evidence for the involvement of amino acid residue Glu398 in the autocatalytic processing of Bacillus licheniformis γ‐glutamyltranspeptidase

Chi¹,

Chen²,

et al. 2012

FEBS Open Bio

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The role of glutamate 398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was explored by site-directed mutagenesis. This glutamate was substituted by either alanine, aspartate, arginine or glutamine and the expressed mutant enzymes were purified to apparent homogeneity with metal-affinity chromatography. SDS–PAGE analysis showed that E398A, E398D and E398K were unable to process themselves into a large and a small subunit. However, E398Q was not only able to process itself, but also had a catalytic activity comparable to that of BlGGT. As compared with the wild-type enzyme, no significant change in circular dichroism spectra was observed for the mutant proteins. Thermal unfolding of BlGGT, E398A, E398D, E398K and E398Q followed the two-state unfolding process with a transition point (Tm) of 47.7–69.4 °C. Tryptophan fluorescence spectra of the mutant enzymes were different from the wild-type protein in terms of fluorescence intensity. Native BlGGT started to unfold beyond ∼1.92 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]0.5, N–U, at 3.07 M equivalent to free energy change (normalΔ⁢GN−UH2⁢normalO) of 14.53 kcal/mol for the N → U process, whereas the denaturation midpoints for the mutant enzymes were 1.31–2.99 M equivalent to Δ⁢GN−UnormalH2⁢O of 3.29–12.05 kcal/mol. Taken together, these results strongly suggest that the explored glutamate residue is indeed important for the autocatalytic processing of BlGGT.

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Biophysical characterization of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases: A comparative analysis

Cited by 23 publications

References 35 publications

Specific biochemical amniotic fluid pattern of fetal isolated esophageal atresia

Specific biochemical amniotic fluid pattern of fetal isolated esophageal atresia

Mutational Analysis of a Highly Conserved PLSSMXP Sequence in the Small Subunit of Bacillus licheniformis γ-Glutamyltranspeptidase

Experimental evidence for the involvement of amino acid residue Glu398 in the autocatalytic processing of Bacillus licheniformis γ‐glutamyltranspeptidase

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