Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-β-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence
Abstract:Metallo-β-lactamases (MBLs) are class B β-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of β-lactam antibiotics. A previous study on BLEG-1 (formerly called Bleg1_2437), a hypothetical protein from Bacillus lehensis G1, revealed sequence similarity and activity to B3 subclass MBLs, despite its evolutionary divergence from these enzymes. Its relatedness to glyoxalase II (GLXII) raises the possibility of its enzymatic promiscuity and unique structural features compare… Show more
“…4 B, Table 3 ). Protein crystals with similar morphology and size (0.3–0.6 mm) were reproducible using the optimized crystallization conditions, which eventually enabled X-ray diffraction and structure determination of BLEG-1 at 1.44 Å (deposited in PDB, ID: 7EV5) [1] . Fig.…”
Section: Methods Validationmentioning
confidence: 96%
“… [16] and Au et al. [1] resulted in less protein aggregation post purification, with the addition of 500 mM NaCl. His-tag cleavage No No Yes Method for buffer exchange, protein concentration and buffer used Ultrafiltration Buffer 3: 10 mM Na 2 HPO 4 •7H 2 O (pH 7).…”
Section: Methods Validationmentioning
confidence: 99%
“…[ 16 ] Au et al. [ 1 ] Remarks Expression host E. coli Rosetta-gami (DE3) E. coli BL21(DE3) E. coli BL21(DE3) Expression plasmid pET-32b pET-28b pET-28b Cultivation medium and volume Luria-Bertani broth 200 mL Luria-Bertani broth 3 L Luria-Bertani broth 3 L Antibiotics in cultivation medium 50 μg/mL ampicillin and 34 μg/mL chloramphenicol 200 mL medium 50 μg/mL kanamycin 3 L medium 50 μg/mL kanamycin 3 L medium 100 µM ZnSO 4 in cultivation medium Absent Present Present 100 µM ZnSO 4 was added to the cultivation medium as a means to allow the target protein to be incorporated with Zn 2+ . Method of purification and buffers used Affinity chromatography Buffer 1: 20 mM Na 2 HPO 4 •7H 2 0, 1 M NaCI (pH 7) Buffer 2: 20 mM Na 2 HPO 4 •7H 2 O, 1 M NaCI, 1 M imidazole (pH 7) Affinity chromatography Buffer 1: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 500 mM NaCl, 50 mM imidazole, 2 mM MgSO 4 (pH 7.4) Buffer 2: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 500 mM NaCl and 500 mM imidazole (pH 7.4) Affinity chromatography Buffer 1: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 500 mM NaCl, 50 mM imidazole, 2 mM MgSO 4 •7H 2 O (pH 7.4) Buffer 2: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 500 mM NaCl, 500 mM imidazole (pH 7.4) Anion exchange chromatography Buffer 3: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 20 mM NaCl, (pH 7.4) Buffer 4: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 500 mM NaCl (pH 7.4) Buffer composition used in Selvaraju et al.…”
Section: Methods Validationmentioning
confidence: 99%
“… The addition of 100 mM NaCl in Buffer 5 (dialysis 2) by Au et al. [1] ensures better buffering of protein for longer duration during crystallization. Protein concentration obtained 3.2 mg 27 mg 32 mg Fig.…”
“…4 B, Table 3 ). Protein crystals with similar morphology and size (0.3–0.6 mm) were reproducible using the optimized crystallization conditions, which eventually enabled X-ray diffraction and structure determination of BLEG-1 at 1.44 Å (deposited in PDB, ID: 7EV5) [1] . Fig.…”
Section: Methods Validationmentioning
confidence: 96%
“… [16] and Au et al. [1] resulted in less protein aggregation post purification, with the addition of 500 mM NaCl. His-tag cleavage No No Yes Method for buffer exchange, protein concentration and buffer used Ultrafiltration Buffer 3: 10 mM Na 2 HPO 4 •7H 2 O (pH 7).…”
Section: Methods Validationmentioning
confidence: 99%
“…[ 16 ] Au et al. [ 1 ] Remarks Expression host E. coli Rosetta-gami (DE3) E. coli BL21(DE3) E. coli BL21(DE3) Expression plasmid pET-32b pET-28b pET-28b Cultivation medium and volume Luria-Bertani broth 200 mL Luria-Bertani broth 3 L Luria-Bertani broth 3 L Antibiotics in cultivation medium 50 μg/mL ampicillin and 34 μg/mL chloramphenicol 200 mL medium 50 μg/mL kanamycin 3 L medium 50 μg/mL kanamycin 3 L medium 100 µM ZnSO 4 in cultivation medium Absent Present Present 100 µM ZnSO 4 was added to the cultivation medium as a means to allow the target protein to be incorporated with Zn 2+ . Method of purification and buffers used Affinity chromatography Buffer 1: 20 mM Na 2 HPO 4 •7H 2 0, 1 M NaCI (pH 7) Buffer 2: 20 mM Na 2 HPO 4 •7H 2 O, 1 M NaCI, 1 M imidazole (pH 7) Affinity chromatography Buffer 1: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 500 mM NaCl, 50 mM imidazole, 2 mM MgSO 4 (pH 7.4) Buffer 2: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 500 mM NaCl and 500 mM imidazole (pH 7.4) Affinity chromatography Buffer 1: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 500 mM NaCl, 50 mM imidazole, 2 mM MgSO 4 •7H 2 O (pH 7.4) Buffer 2: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 500 mM NaCl, 500 mM imidazole (pH 7.4) Anion exchange chromatography Buffer 3: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 20 mM NaCl, (pH 7.4) Buffer 4: 20 mM Na 2 HPO 4 –NaH 2 PO 4 , 500 mM NaCl (pH 7.4) Buffer composition used in Selvaraju et al.…”
Section: Methods Validationmentioning
confidence: 99%
“… The addition of 100 mM NaCl in Buffer 5 (dialysis 2) by Au et al. [1] ensures better buffering of protein for longer duration during crystallization. Protein concentration obtained 3.2 mg 27 mg 32 mg Fig.…”
“…BLEG-1 from Bacillus lehensis G1 alkaliphile is a promiscuous MBL of B3 subclass that can inactivate almost all classes of β-lactam antibiotics except monobactams, and catalyze the hydrolysis of S-D-lactoylglutathione (SLG) (i.e. the substrate of GLXII enzymes) [ 12 , 13 ]. Unlike the bacteria-producing B3 MBLs, GLXII is found in both prokaryotes and eukaryotes.…”
BLEG-1 from Bacillus lehensis G1 is an evolutionary divergent B3 metallo-β-lactamase (MBL) that exhibited both β-lactamase and glyoxalase II (GLXII) activities. Sequence, phylogeny, biochemical and structural relatedness of BLEG-1 to B3 MBL and GLXII suggested BLEG-1 might be an intermediate in the evolutionary path of B3 MBL from GLXII. The unique active site cavity of BLEG-1 that recognizes both β-lactam antibiotics and S-D-lactoylglutathione (SLG) had been postulated as the key factor for its dual activity. In this study, dynamic ensembles of BLEG-1 and its substrate complexes divulged conformational plasticity and binding modes of structurally distinct substrates to the enzyme, providing better insights into its structure-to-function relationship and enzymatic promiscuity. Our results highlight the flexible nature of the active site pocket of BLEG-1, which is governed by concerted loop motions involving loop7+α3+loop8 and loop12 around the catalytic core, thereby moulding the binding pocket and facilitate interactions of BLEG-1 with both ampicillin and SLG. The distribution of (i) predominantly hydrophobic amino acids in the N-terminal domain, and (ii) flexible amino acids with polar and/or charged side chains in both N- and C-termini provide additional advantages to BLEG-1 in confining the aromatic group of ampicillin, and polar groups of SLG, respectively. The importance of these residues for substrates binding was further confirmed by the reduction in MBL and GLXII activities upon alanine substitutions of Ile-10, Phe-57, Arg-94, Leu-95, and Arg-159. Based on molecular dynamics simulation, mutational, and biochemical data presented herein, the catalytic mechanisms of BLEG-1 toward the hydrolysis of β-lactams and SLG were proposed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
