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 Structural basis for carotenoid cleavage by an archaeal carotenoid dioxygenase

2020 ◽  
Vol 117 (33) ◽  
pp. 19914-19925 ◽  
Author(s):  
Anahita Daruwalla ◽  
Jianye Zhang ◽  
Ho Jun Lee ◽  
Nimesh Khadka ◽  
Erik R. Farquhar ◽  
...  
Keyword(s):  
Active Site ◽  
Molecular Basis ◽  
Structural Basis ◽  
Reaction Intermediates ◽  
Catalytic Activities ◽  
Selective Stabilization ◽  
Carotenoid Cleavage Dioxygenases ◽  
Active Site Cavity ◽  
Binding Cleft ◽  
Shed Light

Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD–carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD fromCandidatusNitrosotalea devanaterra (NdCCD).NdCCD was active toward β-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14′–C13′ alkene bond to produce β-apo-14′-carotenals. The structure ofNdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-formingSynechocystisapocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure ofNdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.

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 Mapping the Transglycosylation Relevant Sites of Cold-Adapted β-d-Galactosidase from Arthrobacter sp. 32cB

2020 ◽  
Vol 21 (15) ◽  
pp. 5354
Author(s):  
Maria Rutkiewicz ◽  
Marta Wanarska ◽  
Anna Bujacz
Keyword(s):  
Active Site ◽  
Structural Basis ◽  
Complex Structures ◽  
Loss Of Function ◽  
Significant Drop ◽  
Cold Adapted ◽  
Transglycosylation Reaction ◽  
Structural Details ◽  
Hydrolysis Of ◽  
Shed Light

β-Galactosidase from Arthrobacter sp. 32cB (ArthβDG) is a cold-adapted enzyme able to catalyze hydrolysis of β-d-galactosides and transglycosylation reaction, where galactosyl moiety is being transferred onto an acceptor larger than a water molecule. Mutants of ArthβDG: D207A and E517Q were designed to determine the significance of specific residues and to enable formation of complexes with lactulose and sucrose and to shed light onto the structural basis of the transglycosylation reaction. The catalytic assays proved loss of function mutation E517 into glutamine and a significant drop of activity for mutation of D207 into alanine. Solving crystal structures of two new mutants, and new complex structures of previously presented mutant E441Q enables description of introduced changes within active site of enzyme and determining the importance of mutated residues for active site size and character. Furthermore, usage of mutants with diminished and abolished enzymatic activity enabled solving six complex structures with galactose, lactulose or sucrose bounds. As a result, not only the galactose binding sites were mapped on the enzyme’s surface but also the mode of lactulose, product of transglycosylation reaction, and binding within the enzyme’s active site were determined and the glucopyranose binding site in the distal of active site was discovered. The latter two especially show structural details of transglycosylation, providing valuable information that may be used for engineering of ArthβDG or other analogous galactosidases belonging to GH2 family.

The catalytic mechanism of Pseudomonas aeruginosa cd1 nitrite reductase

2011 ◽  
Vol 39 (1) ◽  
pp. 195-200 ◽  
Author(s):  
Serena Rinaldo ◽  
Giorgio Giardina ◽  
Nicoletta Castiglione ◽  
Valentina Stelitano ◽  
Francesca Cutruzzolà
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Product Inhibition ◽  
Electron Donors ◽  
Nitrite Reduction ◽  
Nitrite Reductases ◽  
Shed Light ◽  
Denitrification Process

The cd1 NiRs (nitrite reductases) are enzymes catalysing the reduction of nitrite to NO (nitric oxide) in the bacterial energy conversion denitrification process. These enzymes contain two distinct redox centres: one covalently bound c-haem, which is reduced by external electron donors, and another peculiar porphyrin, the d1-haem (3,8-dioxo-17-acrylate-porphyrindione), where nitrite is reduced to NO. In the present paper, we summarize the most recent results on the mechanism of nitrite reduction by the cd1 NiR from Pseudomonas aeruginosa. We discuss the essential catalytic features of this enzyme, with special attention to the allosteric regulation of the enzyme's activity and to the mechanism employed to avoid product inhibition, i.e. trapping of the active-site reduced haem by the product NO. These results shed light on the reactivity of cd1 NiRs and assign a central role to the unique d1-haem, present only in this class of enzymes.

scholarly journals Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design

2006 ◽  
Vol 399 (2) ◽  
pp. 199-204 ◽  
Author(s):  
Kamel EL Omari ◽  
Annelies Bronckaers ◽  
Sandra Liekens ◽  
Maria-Jésus Pérez-Pérez ◽  
Jan Balzarini ◽  
...  
Keyword(s):  
Drug Design ◽  
Active Site ◽  
Thymidine Phosphorylase ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Structural Basis ◽  
Closed Conformation ◽  
Ribose Phosphate ◽  
Nucleoside Drugs ◽  
Competitive Product

HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end com-plex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406–415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs.

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.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

2019 ◽  
Vol 476 (6) ◽  
pp. 991-1003 ◽  
Author(s):  
Vijaykumar Pillalamarri ◽  
Tarun Arya ◽  
Neshatul Haque ◽  
Sandeep Chowdary Bala ◽  
Anil Kumar Marapaka ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Covalent Modification ◽  
Structural Basis ◽  
Wild Type ◽  
Methionine Aminopeptidases ◽  
Synthetic Derivative ◽  
Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

scholarly journals Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Matthew J. Belousoff ◽  
Zohar Eyal ◽  
Mazdak Radjainia ◽  
Tofayel Ahmed ◽  
Rebecca S. Bamert ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Structural Information ◽  
Structural Basis ◽  
Common Mechanism ◽  
Resistance Mutations ◽  
Antimicrobial Drugs ◽  
Linezolid Resistance ◽  
Drug Modification ◽  
Antibiotic Resistance Mutations ◽  
Shed Light

ABSTRACT An unorthodox, surprising mechanism of resistance to the antibiotic linezolid was revealed by cryo-electron microscopy (cryo-EM) in the 70S ribosomes from a clinical isolate of Staphylococcus aureus. This high-resolution structural information demonstrated that a single amino acid deletion in ribosomal protein uL3 confers linezolid resistance despite being located 24 Å away from the linezolid binding pocket in the peptidyl-transferase center. The mutation induces a cascade of allosteric structural rearrangements of the rRNA that ultimately results in the alteration of the antibiotic binding site. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance.

scholarly journals Structural basis for an atypical active site of anl-aspartate/glutamate-specific racemase fromEscherichia coli

FEBS Letters ◽  
2015 ◽  
Vol 589 (24PartB) ◽  
pp. 3842-3847 ◽  
Author(s):  
Jae-Woo Ahn ◽  
Jeong Ho Chang ◽  
Kyung-Jin Kim
Keyword(s):  
Active Site ◽  
Structural Basis ◽  
Fromescherichia Coli

scholarly journals Bioreactor and Bioprocess Design Issues in Enzymatic Hydrolysis of Lignocellulosic Biomass

Catalysts ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 680
Author(s):  
Giuseppe Olivieri ◽  
René H. Wijffels ◽  
Antonio Marzocchella ◽  
Maria Elena Russo
Keyword(s):  
Lignocellulosic Biomass ◽  
Enzymatic Saccharification ◽  
Product Inhibition ◽  
Cellulolytic Enzymes ◽  
Main Process ◽  
Bioprocess Design ◽  
Operating Modes ◽  
Biomass Saccharification ◽  
Starting Point ◽  
The Impact

Saccharification of lignocellulosic biomass is a fundamental step in the biorefinery of second generation feedstock. The physicochemical and enzymatic processes for the depolymerization of biomass into simple sugars has been achieved through numerous studies in several disciplines. The present review discusses the development of technologies for enzymatic saccharification in industrial processes. The kinetics of cellulolytic enzymes involved in polysaccharide hydrolysis has been discussed as the starting point for the design of the most promising bioreactor configurations. The main process configurations—proposed so far—for biomass saccharification have been analyzed. Attention was paid to bioreactor configurations, operating modes and possible integrations of this operation within the biorefinery. The focus is on minimizing the effects of product inhibition on enzymes, maximizing yields and concentration of sugars in the hydrolysate, and reducing the impact of enzyme cost on the whole process. The last part of the review is focused on an emerging process based on the catalytic action of laccase applied to lignin depolymerization as an alternative to the consolidated physicochemical pretreatments. The laccases-based oxidative process has been discussed in terms of characteristics that can affect the development of a bioreactor unit where laccases or a laccase-mediator system can be used for biomass delignification.

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