scholarly journals Improving the Substrate Affinity and Catalytic Efficiency of β-Glucosidase Bgl3A from Talaromyces leycettanus JCM12802 by Rational Design

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1882
Author(s):  
Wei Xia ◽  
Yingguo Bai ◽  
Pengjun Shi
Keyword(s):  
Hydrogen Bonding ◽  
Rational Design ◽  
Substrate Binding ◽  
Enzymatic Saccharification ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Glycoside Hydrolases ◽  
Cellulosic Biomass ◽  
Substrate Affinity ◽  
Hydrogen Bonding Networks

Improving the substrate affinity and catalytic efficiency of β-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other β-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower Km values and 1.4–2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other β-glucosidases and even glycoside hydrolases.

scholarly journals Structural and mechanistic basis of the high catalytic activity of monooxygenase Tet(X4) on tigecycline

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Qipeng Cheng ◽  
Yanchu Cheung ◽  
Chenyu Liu ◽  
Qingjie Xiao ◽  
Bo Sun ◽  
...  
Keyword(s):  
Rational Design ◽  
Substrate Binding ◽  
Catalytic Efficiency ◽  
Binding Domain ◽  
Multiple Drug ◽  
High Catalytic Activity ◽  
Multiple Drug Resistant ◽  
Mechanistic Basis ◽  
Substrate Binding Domain ◽  
Fad Binding

Abstract Background Tigecycline is a tetracycline derivative that constitutes one of the last-resort antibiotics used clinically to treat infections caused by both multiple drug-resistant (MDR) Gram-negative and Gram-positive bacteria. Resistance to this drug is often caused by chromosome-encoding mechanisms including over-expression of efflux pumps and ribosome protection. However, a number of variants of the flavin adenine dinucleotide (FAD)-dependent monooxygenase TetX, such as Tet(X4), emerged in recent years as conferring resistance to tigecycline in strains of Enterobacteriaceae, Acinetobacter sp., Pseudomonas sp., and Empedobacter sp. To date, mechanistic details underlying the improvement of catalytic activities of new TetX enzymes are not available. Results In this study, we found that Tet(X4) exhibited higher affinity and catalytic efficiency toward tigecycline when compared to Tet(X2), resulting in the expression of phenotypic tigecycline resistance in E. coli strains bearing the tet(X4) gene. Comparison between the structures of Tet(X4) and Tet(X4)-tigecycline complex and those of Tet(X2) showed that they shared an identical FAD-binding site and that the FAD and tigecycline adopted similar conformation in the catalytic pocket. Although the amino acid changes in Tet(X4) are not pivotal residues for FAD binding and substrate recognition, such substitutions caused the refolding of several alpha helixes and beta sheets in the secondary structure of the substrate-binding domain of Tet(X4), resulting in the formation of a larger number of loops in the structure. These changes in turn render the substrate-binding domain of Tet(X4) more flexible and efficient in capturing substrate molecules, thereby improving catalytic efficiency. Conclusions Our works provide a better understanding of the molecular recognition of tigecycline by the TetX enzymes; these findings can help guide the rational design of the next-generation tetracycline antibiotics that can resist inactivation of the TetX variants.

Improved catalytic activity and stability of cellobiohydrolase (Cel6A) from the Aspergillus fumigatus by rational design

2020 ◽  
Vol 33 ◽  
Author(s):  
Subba Reddy Dodda ◽  
Nibedita Sarkar ◽  
Piyush Jain ◽  
Kaustav Aikat ◽  
Sudit S Mukhopadhyay
Keyword(s):  
Catalytic Activity ◽  
Protein Engineering ◽  
Aspergillus Fumigatus ◽  
Rational Design ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Substrate Affinity ◽  
Wild Type ◽  
Dynamics Simulations ◽  
Cellulose Binding

Abstract Cheap production of glucose is the current challenge for the production of cheap bioethanol. Ideal protein engineering approaches are required for improving the efficiency of the members of the cellulase, the enzyme complex involved in the saccharification process of cellulose. An attempt was made to improve the efficiency of the cellobiohydrolase (Cel6A), the important member of the cellulase isolated from Aspergillus fumigatus (AfCel6A). Structure-based variants of AfCel6A were designed. Amino acids surrounding the catalytic site and conserved residues in the cellulose-binding domain were targeted (N449V, N168G, Y50W and W24YW32Y). I mutant 3 server was used to identify the potential variants based on the free energy values (∆∆G). In silico structural analyses and molecular dynamics simulations evaluated the potentiality of the variants for increasing thermostability and catalytic activity of Cel6A. Further enzyme studies with purified protein identified the N449V is highly thermo stable (60°C) and pH tolerant (pH 5–7). Kinetic studies with Avicel determined that substrate affinity of N449V (Km =0.90 ± 0.02) is higher than the wild type (1.17 ± 0.04) and the catalytic efficiency (Kcat/Km) of N449V is ~2-fold higher than wild type. All these results suggested that our strategy for the development of recombinant enzyme is a right approach for protein engineering.

scholarly journals Comparative enzymology of 11β-hydroxysteroid dehydrogenase type 1 from six species

2005 ◽  
Vol 35 (1) ◽  
pp. 89-101 ◽  
Author(s):  
Spyridon Arampatzis ◽  
Bert Kadereit ◽  
Daniela Schuster ◽  
Zoltan Balazs ◽  
Roberto A S Schweizer ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Animal Experiments ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Binding Pocket ◽  
Substrate Affinity ◽  
Drug Candidates ◽  
Species Specific

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1), catalyzing the intracellular activation of cortisone to cortisol, is currently considered a promising target to treat patients with metabolic syndrome; hence, there is considerable interest in the development of selective inhibitors. For preclinical tests of such inhibitors, the characteristics of 11β-HSD1 from the commonly used species have to be known. Therefore, we determined differences in substrate affinity and inhibitor effects for 11β-HSD1 from six species. The differences in catalytic activities with cortisone and 11-dehydrocorticosterone were rather modest. Human, hamster and guinea-pig 11β-HSD1 displayed the highest catalytic efficiency in the oxoreduction of cortisone, while mouse and rat showed intermediate and dog the lowest activity. Murine 11β-HSD1 most efficiently reduced 11-dehydrocorticosterone, while the enzyme from dog showed lower activity than those from the other species. 7-Ketocholesterol (7KC) was stereospecifically converted to 7β-hydroxycholesterol by recombinant 11β-HSD1 from all species analyzed except hamster, which showed a slight preference for the formation of 7α-hydroxycholesterol. Importantly, guinea-pig and canine 11β-HSD1 displayed very low 7-oxoreductase activities. Furthermore, we demonstrate significant species-specific variability in the potency of various 11β-HSD1 inhibitors, including endogenous compounds, natural chemicals and pharmaceutical compounds. The results suggest significant differences in the three-dimensional organization of the hydrophobic substrate-binding pocket of 11β-HSD1, and they emphasize that species-specific variability must be considered in the interpretation of results obtained from different animal experiments. The assessment of such differences, by cell-based test systems, may help to choose the appropriate animal for safety and efficacy studies of novel potential drug candidates.

scholarly journals Catalytic Efficiency of Basidiomycete Laccases: Redox Potential versus Substrate-Binding Pocket Structure

Catalysts ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 152 ◽  
Author(s):  
Olga Glazunova ◽  
Nikita Trushkin ◽  
Konstantin Moiseenko ◽  
Ivan Filimonov ◽  
Tatyana Fedorova
Keyword(s):  
Redox Potential ◽  
Substrate Binding ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Structural Features ◽  
Redox Potentials ◽  
Substrate Binding Pocket ◽  
Trametes Hirsuta ◽  
Special Cases ◽  
Almost All

Laccases are copper-containing oxidases that catalyze a one-electron abstraction from various phenolic and non-phenolic compounds with concomitant reduction of molecular oxygen to water. It is well-known that laccases from various sources have different substrate specificities, but it is not completely clear what exactly provides these differences. The purpose of this work was to study the features of the substrate specificity of four laccases from basidiomycete fungi Trametes hirsuta, Coriolopsis caperata, Antrodiella faginea, and Steccherinum murashkinskyi, which have different redox potentials of the T1 copper center and a different structure of substrate-binding pockets. Enzyme activity toward 20 monophenolic substances and 4 phenolic dyes was measured spectrophotometrically. The kinetic parameters of oxidation of four lignans and lignan-like substrates were determined by monitoring of the oxygen consumption. For the oxidation of the high redox potential (>700 mV) monophenolic substrates and almost all large substrates, such as phenolic dyes and lignans, the redox potential difference between the enzyme and the substrate (ΔE) played the defining role. For the low redox potential monophenolic substrates, ΔE did not directly influence the laccase activity. Also, in the special cases, the structure of the large substrates, such as dyes and lignans, as well as some structural features of the laccases (flexibility of the substrate-binding pocket loops and some amino acid residues in the key positions) affected the resulting catalytic efficiency.

scholarly journals A kinase bioscavenger provides antibiotic resistance by extremely tight substrate binding

2020 ◽  
Vol 6 (26) ◽  
pp. eaaz9861 ◽  
Author(s):  
Stanislav S. Terekhov ◽  
Yuliana A. Mokrushina ◽  
Anton S. Nazarov ◽  
Alexander Zlobin ◽  
Arthur Zalevsky ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Microbial Communities ◽  
Active Site ◽  
Substrate Binding ◽  
Catalytic Efficiency ◽  
Crystallographic Analysis ◽  
Diffusion Limit ◽  
Substrate Affinity ◽  
Multiscale Simulations ◽  
Substrate Promiscuity

Microbial communities are self-controlled by repertoires of lethal agents, the antibiotics. In their turn, these antibiotics are regulated by bioscavengers that are selected in the course of evolution. Kinase-mediated phosphorylation represents one of the general strategies for the emergence of antibiotic resistance. A new subfamily of AmiN-like kinases, isolated from the Siberian bear microbiome, inactivates antibiotic amicoumacin by phosphorylation. The nanomolar substrate affinity defines AmiN as a phosphotransferase with a unique catalytic efficiency proximal to the diffusion limit. Crystallographic analysis and multiscale simulations revealed a catalytically perfect mechanism providing phosphorylation exclusively in the case of a closed active site that counteracts substrate promiscuity. AmiN kinase is a member of the previously unknown subfamily representing the first evidence of a specialized phosphotransferase bioscavenger.

scholarly journals Site-directed Mutagenesis of a β-Glycoside Hydrolase fromLentinula edodes

Molecules ◽  
2018 ◽  
Vol 24 (1) ◽  
pp. 59 ◽  
Author(s):  
Jing-Jing Chen ◽  
Xiao Liang ◽  
Tian-Jiao Chen ◽  
Jin-Ling Yang ◽  
Ping Zhu
Keyword(s):  
Glycoside Hydrolase ◽  
Lentinula Edodes ◽  
Catalytic Efficiency ◽  
Structure And Function ◽  
Glycoside Hydrolases ◽  
Site Directed Mutagenesis ◽  
Substrate Affinity ◽  
And Function ◽  
The Relationship ◽  
Loop Conformation

The β-glycoside hydrolases (LXYL-P1−1 and LXYL-P1−2) from Lentinula edodes (strain M95.33) can specifically hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) to form 10-deacetyltaxol for the semi-synthesis of Taxol. Our previous study showed that both the I368T mutation in LXYL-P1−1 and the T368E mutation in LXYL-P1−2 could increase the enzyme activity, which prompted us to investigate the effect of the I368E mutation on LXYL-P1−1 activity. In this study, the β-xylosidase and β-glucosidase activities of LXYL-P1−1I368E were 1.5 and 2.2 times higher than those of LXYL-P1−1. Most importantly, combination of I368E and V91S exerted the cumulative effects on the improvement of the enzyme activities and catalytic efficiency. The β-xylosidase and β-glucosidase activities of the double mutant LXYL-P1−1V91S/I368E were 3.2 and 1.7-fold higher than those of LXYL-P1−1I368E. Similarly, the catalytic efficiency of LXYL-P1−1V91S/I368E on 7-β-xylosyl-10-deacetyltaxol was 1.8-fold higher than that of LXYL-P1−1I368E due to the dramatic increase in the substrate affinity. Molecular docking results suggest that the V91S and I368E mutation might positively promote the interaction between enzyme and substrate through altering the loop conformation near XDT and increasing the hydrogen bonds among Ser91, Trp301, and XDT. This study lays the foundation for exploring the relationship between the structure and function of the β-glycoside hydrolases.

Understanding substrate binding and the role of gatekeeping residues in PigC access tunnels

2021 ◽  
Author(s):  
Stefanie Brands ◽  
Jarno G. Sikkens ◽  
Mehdi D. Davari ◽  
Hannah U. C. Brass ◽  
Andreas S. Klein ◽  
...  
Keyword(s):  
Rational Design ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Short Chain ◽  
Substrate Binding Pocket

Prodigiosin ligase PigC has been engineered by semi-rational design to accept short chain-pyrroles, providing molecular understanding of access tunnels and the substrate-binding pocket.

scholarly journals ω-Oxidation impairs oxidizability of polyenoic fatty acids by 15-lipoxygenases: consequences for substrate orientation at the active site

1998 ◽  
Vol 336 (2) ◽  
pp. 345-352 ◽  
Author(s):  
Igor IVANOV ◽  
Kristin SCHWARZ ◽  
Herman G. HOLZHÜTTER ◽  
Galina MYAGKOVA ◽  
Hartmut KÜHN
Keyword(s):  
Fatty Acids ◽  
Active Site ◽  
Substrate Binding ◽  
Salt Bridge ◽  
Binding Pocket ◽  
Substrate Affinity ◽  
Carboxylate Group ◽  
Fatty Acid Binding ◽  
Substrate Orientation ◽  
Binding Cleft

During oxygenation by 15-lipoxygenases, polyenoic fatty acids are bound at the active site in such a way that the ω-terminus of the fatty acids penetrates into the substrate binding pocket. In contrast, for arachidonic acid 5-lipoxygenation, an inverse head to tail orientation has been suggested. However, an inverse orientation may be hindered by the large energy barrier associated with burying the charged carboxylate group in the hydrophobic environment of the substrate binding cleft. We studied the oxygenation kinetics of ω-modified fatty acids by 15-lipoxygenases and found that ω-hydroxylation strongly impaired substrate affinity (higher Km), but only moderately altered Vmax. In contrast, ω-carboxylation completely prevented the lipoxygenase reaction; however, methylation of the additional carboxylate group restored the activity. Arg403 of the human 15-lipoxygenase has been implicated in fatty acid binding by forming a salt bridge with the carboxylate group, and thus mutation of this amino acid to an uncharged residue was supposed to favour an inverse substrate orientation. The prepared Arg403 → Leu mutant of the rabbit 15-lipoxygenase was found to be a less effective catalyst of linoleic acid oxygenation. However, the oxygenation rate of ω-hydroxyarachidonic acid was similar when the wild-type and mutant enzyme were compared, and the patterns of oxygenation products were identical for both enzyme species. These data suggest that introduction of a polar, or even charged residue, at the ω-terminus of substrate fatty acids in connection with mutation of Arg403 may not alter substrate alignment at the active site of 15-lipoxygenases.

Hierarchy of π-Stacking Determines the Conformational Preference of Bis-Squaraines

2020 ◽  
Author(s):  
Abhishek Singh ◽  
Reman K. Singh ◽  
G Naresh Patwari
Keyword(s):  
Hydrogen Bonding ◽  
Rational Design ◽  
Biological Molecules ◽  
Conformational Space ◽  
X Ray Crystallography ◽  
Simple Strategy ◽  
Induced Folding ◽  
Π Stacking ◽  
Varying Length ◽  
Dynamics Simulations

The rational design of conformationally controlled foldable modules can lead to a deeper insight into the conformational space of complex biological molecules where non-covalent interactions such as hydrogen bonding and π-stacking are known to play a pivotal role. Squaramides are known to have excellent hydrogen bonding capabilities and hence, are ideal molecules for designing foldable modules that can mimic the secondary structures of bio-molecules. The π-stacking induced folding of bis-squaraines tethered using aliphatic primary and secondary-diamine linkers of varying length is explored with a simple strategy of invoking small perturbations involving the length linkers and degree of substitution. Solution phase NMR investigations in combination with molecular dynamics simulations suggest that bis-squaraines predominantly exist as extended conformations. Structures elucidated by X-ray crystallography confirmed a variety of folded and extended secondary conformations including hairpin turns and 𝛽-sheets which are determined by the hierarchy of π-stacking relative to N–H···O hydrogen bonds.

Hierarchy of π-Stacking Determines the Conformational Preference of Bis-Squaraines

2020 ◽  
Author(s):  
Abhishek Singh ◽  
Reman K. Singh ◽  
G Naresh Patwari
Keyword(s):  
Hydrogen Bonding ◽  
Rational Design ◽  
Biological Molecules ◽  
Conformational Space ◽  
X Ray Crystallography ◽  
Simple Strategy ◽  
Induced Folding ◽  
Π Stacking ◽  
Varying Length ◽  
Dynamics Simulations

The rational design of conformationally controlled foldable modules can lead to a deeper insight into the conformational space of complex biological molecules where non-covalent interactions such as hydrogen bonding and π-stacking are known to play a pivotal role. Squaramides are known to have excellent hydrogen bonding capabilities and hence, are ideal molecules for designing foldable modules that can mimic the secondary structures of bio-molecules. The π-stacking induced folding of bis-squaraines tethered using aliphatic primary and secondary-diamine linkers of varying length is explored with a simple strategy of invoking small perturbations involving the length linkers and degree of substitution. Solution phase NMR investigations in combination with molecular dynamics simulations suggest that bis-squaraines predominantly exist as extended conformations. Structures elucidated by X-ray crystallography confirmed a variety of folded and extended secondary conformations including hairpin turns and 𝛽-sheets which are determined by the hierarchy of π-stacking relative to N–H···O hydrogen bonds.

Sign in / Sign up

Export Citation Format

Share Document