scholarly journals Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

2019 ◽  
Author(s):  
Sushant K Sinha ◽  
Shibashis Das ◽  
Sukanya Konar ◽  
Pradip Kr. Ghorai ◽  
Rahul Das ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Active Site ◽  
Specific Activity ◽  
Gene Encoding ◽  
Conserved Residues ◽  
Active Site Pocket ◽  
Glucose Inhibition ◽  
Glucose Tolerant ◽  
Dynamics Simulations ◽  
Hydrolysis Of

Abstractβ-glucosidase catalyzes the hydrolysis of β-1,4 linkage between two glucose molecules in cello-oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition of β-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with β-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase in Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity stimulated in the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and the β-glucosidase at lower concentrations of glucose that however does not lead to enzyme inhibition. We identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced substrate accessibility and significant improvement in activity. Analysis of structures and in combination with molecular dynamics simulations show that glucose increases the accessibility of the substrate by enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific activity up to 0.5 M glucose. Such novel regulation of β-glucosidase activity by its reaction product may offer novel ways of engineering glucose tolerance.

scholarly journals Structural basis for glucose tolerance in GH1 β-glucosidases

2014 ◽  
Vol 70 (6) ◽  
pp. 1631-1639 ◽  
Author(s):  
Priscila Oliveira de Giuseppe ◽  
Tatiana de Arruda Campos Brasil Souza ◽  
Flavio Henrique Moreira Souza ◽  
Leticia Maria Zanphorlin ◽  
Carla Botelho Machado ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Active Site ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Product Inhibition ◽  
Structural Basis ◽  
Clear Correlation ◽  
Biomass Saccharification ◽  
Glucose Tolerant ◽  
Shed Light

Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some β-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal β-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the −1 subsite. The GH1 family β-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification.

scholarly journals High-resolution crystal structure of a polyextreme GH43 glycosidase fromHalothermothrix oreniiwith α-L-arabinofuranosidase activity

2015 ◽  
Vol 71 (3) ◽  
pp. 338-345 ◽  
Author(s):  
Noor Hassan ◽  
Lokesh D. Kori ◽  
Rosaria Gandini ◽  
Bharat K. C. Patel ◽  
Christina Divne ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Thermal Stability ◽  
Active Site ◽  
Metal Binding ◽  
Marine Bacterium ◽  
Specific Activity ◽  
Divalent Cations ◽  
Molecular Replacement ◽  
Gene Encoding ◽  
Hydrophobic Amino Acids

A gene from the heterotrophic, halothermophilic marine bacteriumHalothermothrix oreniihas been cloned and overexpressed inEscherichia coli. This gene encodes the only glycoside hydrolase of family 43 (GH43) produced byH. orenii. The crystal structure of theH. oreniiglycosidase was determined by molecular replacement and refined at 1.10 Å resolution. As for other GH43 members, the enzyme folds as a five-bladed β-propeller. The structure features a metal-binding site on the propeller axis, near the active site. Based on thermal denaturation data, theH. oreniiglycosidase depends on divalent cations in combination with high salt for optimal thermal stability against unfolding. A maximum melting temperature of 76°C was observed in the presence of 4 MNaCl and Mn2+at pH 6.5. The gene encoding theH. oreniiGH43 enzyme has previously been annotated as a putative α-L-arabinofuranosidase. Activity was detected withp-nitrophenyl-α-L-arabinofuranoside as a substrate, and therefore the nameHoAraf43 was suggested for the enzyme. In agreement with the conditions for optimal thermal stability against unfolding, the highest arabinofuranosidase activity was obtained in the presence of 4 MNaCl and Mn2+at pH 6.5, giving a specific activity of 20–36 µmol min−1 mg−1. The active site is structurally distinct from those of other GH43 members, including arabinanases, arabinofuranosidases and xylanases. This probably reflects the special requirements for degrading the unique biomass available in highly saline aqueous ecosystems, such as halophilic algae and halophytes. The amino-acid distribution ofHoAraf43 has similarities to those of mesophiles, thermophiles and halophiles, but also has unique features, for example more hydrophobic amino acids on the surface and fewer buried charged residues.

Inhibition of α-amylase Activity by Zn2+: Insights from Spectroscopy and Molecular Dynamics Simulations

2019 ◽  
Vol 15 (5) ◽  
pp. 510-520 ◽  
Author(s):  
Si-Ming Liao ◽  
Nai-Kun Shen ◽  
Ge Liang ◽  
Bo Lu ◽  
Zhi-Long Lu ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Molecular Dynamics ◽  
Blood Glucose ◽  
Molecular Dynamics Simulations ◽  
Dihedral Angle ◽  
Active Site ◽  
Amylase Activity ◽  
Coordination Interaction ◽  
Active Site Pocket ◽  
Dynamics Simulations

Background:Inhibition of α-amylase activity is an important strategy in the treatment of diabetes mellitus. An important treatment for diabetes mellitus is to reduce the digestion of carbohydrates and blood glucose concentrations. Inhibiting the activity of carbohydrate-degrading enzymes such as α-amylase and glucosidase significantly decreases the blood glucose level. Most inhibitors of α-amylase have serious adverse effects, and the α-amylase inactivation mechanisms for the design of safer inhibitors are yet to be revealed.Objective:In this study, we focused on the inhibitory effect of Zn2+ on the structure and dynamic characteristics of α-amylase from Anoxybacillus sp. GXS-BL (AGXA), which shares the same catalytic residues and similar structures as human pancreatic and salivary α-amylase (HPA and HSA, respectively).Methods:Circular dichroism (CD) spectra of the protein (AGXA) in the absence and presence of Zn2+ were recorded on a Chirascan instrument. The content of different secondary structures of AGXA in the absence and presence of Zn2+ was analyzed using the online SELCON3 program. An AGXA amino acid sequence similarity search was performed on the BLAST online server to find the most similar protein sequence to use as a template for homology modeling. The pocket volume measurer (POVME) program 3.0 was applied to calculate the active site pocket shape and volume, and molecular dynamics simulations were performed with the Amber14 software package.Results:According to circular dichroism experiments, upon Zn2+ binding, the protein secondary structure changed obviously, with the α-helix content decreasing and β-sheet, β-turn and randomcoil content increasing. The structural model of AGXA showed that His217 was near the active site pocket and that Phe178 was at the outer rim of the pocket. Based on the molecular dynamics trajectories, in the free AGXA model, the dihedral angle of C-CA-CB-CG displayed both acute and planar orientations, which corresponded to the open and closed states of the active site pocket, respectively. In the AGXA-Zn model, the dihedral angle of C-CA-CB-CG only showed the planar orientation. As Zn2+ was introduced, the metal center formed a coordination interaction with H217, a cation-π interaction with W244, a coordination interaction with E242 and a cation-π interaction with F178, which prevented F178 from easily rotating to the open state and inhibited the activity of the enzyme.Conclusion:This research may have uncovered a subtle mechanism for inhibiting the activity of α-amylase with transition metal ions, and this finding will help to design more potent and specific inhibitors of α-amylases.

scholarly journals Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance

2020 ◽  
Vol 295 (21) ◽  
pp. 7376-7390
Author(s):  
Cameron A. Brown ◽  
Liya Hu ◽  
Zhizeng Sun ◽  
Meha P. Patel ◽  
Sukrit Singh ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
X Ray ◽  
X Ray Crystallography ◽  
Naturally Occurring ◽  
Bacterial Drug Resistance ◽  
Evolutionary Trajectories ◽  
Dynamics Simulations ◽  
Negative Epistasis ◽  
Hydrolysis Of

CTX-M β-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-μs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.

scholarly journals Effect of A22 on the Conformation of Bacterial Actin MreB

2019 ◽  
Vol 20 (6) ◽  
pp. 1304 ◽  
Author(s):  
Elvis Awuni ◽  
Yuguang Mu
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Conformational Change ◽  
Active Site ◽  
Structural Changes ◽  
Dynamics Simulations ◽  
Hydrolysis Of ◽  
Insight Into

The mechanism of the antibiotic molecule A22 is yet to be clearly understood. In a previous study, we carried out molecular dynamics simulations of a monomer of the bacterial actin-like MreB in complex with different nucleotides and A22, and suggested that A22 impedes the release of Pi from the active site of MreB after the hydrolysis of ATP, resulting in filament instability. On the basis of the suggestion that Pi release occurs on a similar timescale to polymerization and that polymerization can occur in the absence of nucleotides, we sought in this study to investigate a hypothesis that A22 impedes the conformational change in MreB that is required for polymerization through molecular dynamics simulations of the MreB protofilament in the apo, ATP+, and ATP-A22+ states. We suggest that A22 inhibits MreB in part by antagonizing the ATP-induced structural changes required for polymerization. Our data give further insight into the polymerization/depolymerization dynamics of MreB and the mechanism of A22.

scholarly journals Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

2014 ◽  
Vol 58 (10) ◽  
pp. 5994-6002 ◽  
Author(s):  
Melina Ruggiero ◽  
Frédéric Kerff ◽  
Raphaël Herman ◽  
Frédéric Sapunaric ◽  
Moreno Galleni ◽  
...  
Keyword(s):  
Active Site ◽  
Amino Acid Identity ◽  
Conserved Residues ◽  
Class A ◽  
Extended Spectrum ◽  
Active Site Cavity ◽  
Bond Network ◽  
Hydrolysis Of ◽  
Structure And Activity

ABSTRACTPER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singulartransbond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.

scholarly journals Structural insights into the mechanism of the drastic changes in enzymatic activity of the cytochrome P450 vitamin D3hydroxylase (CYP107BR1) caused by a mutation distant from the active site

2017 ◽  
Vol 73 (5) ◽  
pp. 266-275 ◽  
Author(s):  
Yoshiaki Yasutake ◽  
Tomoshi Kameda ◽  
Tomohiro Tamura
Keyword(s):  
Vitamin D ◽  
Hydrogen Bonds ◽  
Active Site ◽  
Structural Changes ◽  
Conformational Transition ◽  
Specific Activity ◽  
Cytochromes P450 ◽  
Site Directed Mutagenesis ◽  
Highly Active ◽  
Dynamics Simulations

Cytochromes P450 (P450s) are haem-containing enzymes that catalyze medically and industrially important oxidative reactions, and many P450s have been subjected to directed evolution and site-directed mutagenesis to improve their activity and substrate specificity. Nonetheless, in most cases the mechanism that leads to drastic changes in specific activity after the introduction of an amino-acid substitution distant from the active-site pocket is unclear. Here, two crystal structures of inactive mutants of the P450 vitamin D3hydroxylase (Vdh), Vdh-F106V and Vdh-L348M, which were obtained in the course of protein-engineering experiments on Vdh, are reported. The overall structures of these mutants show an open conformation similar to that of wild-type Vdh (Vdh-WT), whereas a rearrangement of the common main-chain hydrogen bonds is observed in the CD-loop (residues 102–106), resulting in a more compactly folded CD-loop relative to that of Vdh-WT. The previously reported structures of Vdh-WT and of the highly active Vdh-T107A and Vdh-K1 mutants have a more stretched CD-loop, with partial formation of 310-helix-type hydrogen bonds, both in the open and closed states. Molecular-dynamics simulations also showed that the frequency of the 310-helix is significantly reduced in Vdh-F106V and Vdh-L348M. The closed conformation is crucial for substrate and ferredoxin binding to initiate the catalytic reaction of Vdh. Therefore, it is implied that the small local structural changes observed in this study might disrupt the conformational transition from the open to the closed state, thereby leading to a complete loss of vitamin D3hydroxylase activity.

scholarly journals Heterologous Expression in Pichia pastoris and Characterization of an Endogenous Thermostable and High-Glucose-Tolerant β-Glucosidase from the Termite Nasutitermes takasagoensis

2012 ◽  
Vol 78 (12) ◽  
pp. 4288-4293 ◽  
Author(s):  
Cristiane Akemi Uchima ◽  
Gaku Tokuda ◽  
Hirofumi Watanabe ◽  
Katsuhiko Kitamoto ◽  
Manabu Arioka
Keyword(s):  
Glucose Tolerance ◽  
Biomass Conversion ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Glycoside Hydrolase Family ◽  
Potential Candidate ◽  
Native Form ◽  
Content Type ◽  
Glucose Inhibition ◽  
Glucose Tolerant

ABSTRACTTermites are well-known cellulose decomposers and can give researchers insights into how to utilize lignocellulosic biomass in the actual scenario of energy consumption. In this work, an endogenous β-glucosidase from the midgut of the higher termiteNasutitermes takasagoensiswas purified to homogeneity by Ni2+affinity chromatography and its properties were characterized. This β-glucosidase (G1mgNtBG1), which belongs to glycoside hydrolase family 1, is a homotrimer in its native form, with a molecular mass of 169.5 kDa, as demonstrated by gel filtration chromatography. The enzyme displayed maximum activity at pH 5.5 and had broad substrate specificities toward several saccharides, including cellobiose. G1mgNtBG1 showed a relatively high temperature optimum of 65°C and one of the highest levels of glucose tolerance among several β-glucosidases already characterized, with aKiof 600 mM glucose. To examine the applicability of G1mgNtBG1 in biomass conversion, we compared the thermostability and glucose tolerance of G1mgNtBG1 with those of Novozym 188. We found that G1mgNtBG1 was more thermostable after 5 h of incubation at 60°C and more resistant to glucose inhibition than Novozym 188. Furthermore, our result suggests that G1mgNtBG1 acts synergistically with Celluclast 1.5 L in releasing reducing sugars from Avicel. Thus, G1mgNtBG1 seems to be a potential candidate for use as a supplement in the hydrolysis of biomass.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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