scholarly journals Structural insights into the putative bacterial acetylcholinesterase ChoE and its substrate inhibition mechanism

2020 ◽  
Vol 295 (26) ◽  
pp. 8708-8724
Author(s):  
Van Dung Pham ◽  
Tuan Anh To ◽  
Cynthia Gagné-Thivierge ◽  
Manon Couture ◽  
Patrick Lagüe ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Substrate Inhibition ◽  
Catalytic Triad ◽  
Inhibition Mechanism ◽  
Structural Determinants ◽  
Mutant Proteins ◽  
Carbon And Nitrogen ◽  
Structural Insights ◽  
Substrate Concentrations

Mammalian acetylcholinesterase (AChE) is well-studied, being important in both cholinergic brain synapses and the peripheral nervous systems and also a key drug target for many diseases. In contrast, little is known about the structures and molecular mechanism of prokaryotic acetylcholinesterases. We report here the structural and biochemical characterization of ChoE, a putative bacterial acetylcholinesterase from Pseudomonas aeruginosa. Analysis of WT and mutant strains indicated that ChoE is indispensable for P. aeruginosa growth with acetylcholine as the sole carbon and nitrogen source. The crystal structure of ChoE at 1.35 Å resolution revealed that this enzyme adopts a typical fold of the SGNH hydrolase family. Although ChoE and eukaryotic AChEs catalyze the same reaction, their overall structures bear no similarities constituting an interesting example of convergent evolution. Among Ser-38, Asp-285, and His-288 of the catalytic triad residues, only Asp-285 was not essential for ChoE activity. Combined with kinetic analyses of WT and mutant proteins, multiple crystal structures of ChoE complexed with substrates, products, or reaction intermediate revealed the structural determinants for substrate recognition, snapshots of the various catalytic steps, and the molecular basis of substrate inhibition at high substrate concentrations. Our results indicate that substrate inhibition in ChoE is due to acetate release being blocked by the binding of a substrate molecule in a nonproductive mode. Because of the distinct overall folds and significant differences of the active site between ChoE and eukaryotic AChEs, these structures will serve as a prototype for other prokaryotic acetylcholinesterases.

scholarly journals Biophysical and Biochemical Characterization of Avian Secretory Component Provides Structural Insights into the Evolution of the Polymeric Ig Receptor

2016 ◽  
Vol 197 (4) ◽  
pp. 1408-1414 ◽  
Author(s):  
Beth M. Stadtmueller ◽  
Zhongyu Yang ◽  
Kathryn E. Huey-Tubman ◽  
Helena Roberts-Mataric ◽  
Wayne L. Hubbell ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Secretory Component ◽  
Structural Insights

scholarly journals Structural and Biochemical Characterization of an Active ArylamineN-Acetyltransferase Possessing a Non-canonical Cys-His-Glu Catalytic Triad

2013 ◽  
Vol 288 (31) ◽  
pp. 22493-22505 ◽  
Author(s):  
Xavier Kubiak ◽  
Inès Li de la Sierra-Gallay ◽  
Alain F. Chaffotte ◽  
Benjamin Pluvinage ◽  
Patrick Weber ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Triad

scholarly journals Biochemical characterization of the methylmercaptopropionate:cob(I)alamin methyltransferase fromMethanosarcina acetivorans

2019 ◽  
Author(s):  
He Fu ◽  
Michelle N. Goettge ◽  
William W. Metcalf
Keyword(s):  
Transfer Reaction ◽  
Biochemical Characterization ◽  
Detailed Knowledge ◽  
Methanosarcina Acetivorans ◽  
Coenzyme M ◽  
Metabolic Intermediate ◽  
High Substrate ◽  
Methyl Transfer ◽  
Substrate Concentrations

ABSTRACTMethanogenesis from methylated substrates is initiated by substrate specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, theMethanosarcina acetivoransMtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate(MMPA):Coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, withkcatof 0.315 s-1and apparentKmfor MPA of 12 μM. CoM did not serve as a methyl acceptor, thus a second, unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, could also be demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were co-expressed. Biochemical characterization of this complex was not feasible because the corrinoid cofactor of this complex was in the inactive Co(II) state and could not be reactivated by incubation with strong reductants. The MtsF protein, comprised of both corrinoid and methyltransferase domains, co-purifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism.IMPORTANCEMMPA is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate methyl-CoM. Therefore, additional enzymes required for methanogenic MMPA catabolism await discovery.

scholarly journals Inhibition Effect of Ca2+ Ions on Sucrose Hydrolysis Using Invertase

2019 ◽  
Vol 14 (3) ◽  
pp. 646 ◽  
Author(s):  
Hargono Hargono ◽  
Bakti Jos ◽  
Abdullah Abdullah ◽  
Teguh Riyanto
Keyword(s):  
Kinetic Parameters ◽  
Competitive Inhibition ◽  
Substrate Inhibition ◽  
Competitive Inhibitor ◽  
Inhibition Mechanism ◽  
Fermentable Sugar ◽  
Sucrose Hydrolysis ◽  
Substrate Concentrations ◽  
High Sucrose ◽  
Burk Plot

Fermentable sugar for bioethanol production can be produced from molasses due to its high sucrose content but Ca2+ ions found in the molasses may affect the hydrolysis. Therefore, this paper was focused to study the effect of Ca2+ ions as CaO on sucrose hydrolysis using invertase and to obtain the kinetic parameters. The kinetic parameters (KM and Vmax) were obtained using a Lineweaver-Burk plot. The value of KM and Vmax parameters were 36.181 g/L and 21.322 g/L.h, respectively. The Ca2+ ions act as competitive inhibitor in sucrose hydrolysis using invertase. Therefore, the inhibition mechanism was followed the competitive inhibition mechanism. The value of inhibition constant was 0.833 g/g. These parameters were obtained from the non-substrate inhibition process because this study used the low substrate concentrations which means the fermentable sugar production was low. Hence, there were still more challenges to studying the simultaneous effect of substrate and Ca2+ on sucrose hydrolysis to produce high fermentable sugar. Copyright © 2019 BCREC Group. All rights reserved 

scholarly journals Biochemical Characterization of Gyp6p, a Ypt/Rab-specific GTPase-activating Protein from Yeast

2001 ◽  
Vol 276 (15) ◽  
pp. 12135-12139 ◽  
Author(s):  
Elke Will ◽  
Dieter Gallwitz
Keyword(s):  
Biochemical Characterization ◽  
Three Dimensional ◽  
Significant Inhibition ◽  
Dimensional Structure ◽  
Physical Interaction ◽  
Mutant Proteins ◽  
Gtpase Activating Proteins ◽  
Two Hybrid ◽  
Intrinsic Gtpase Activity

Gyp6p from yeast belongs to the GYP family of Ypt/Rab-specific GTPase-activating proteins, and Ypt6p is its preferred substrate (Strom, M., Vollmer, P., Tan, T. J., and Gallwitz, D. (1993)Nature361, 736–739). We have investigated the kinetic parameters of Gyp6p/Ypt6p interactions and find that Gyp6p accelerates the intrinsic GTPase activity of Ypt6p (0.0002 min−1) by a factor of 5 × 106and that they have a very low affinity for its preferred substrate(Km= 592 μm). Substitution with alanine of several arginines, which Gyp6p shares with other GYP family members, resulted in significant inhibition of GAP activity. Replacement of arginine-155 with either alanine or lysine abolished its GAP activity, indicating a direct involvement of this strictly conserved arginine in catalysis. Physical interaction of the catalytically inactive Gyp6(R155A) mutant GAP with Ypt6 wild-type and Ypt6 mutant proteins could be demonstrated with the two-hybrid system. Short N-terminal and C-terminal truncations of Gyp6p resulted in a complete loss of GAP activity and Ypt6p binding, showing that in contrast to two other Gyp proteins studied previously, most of the 458 amino acid-long Gyp6p sequence is required to form a three-dimensional structure that allows substrate binding and catalysis.

scholarly journals Biochemical Characterization of the Methylmercaptopropionate:Cob(I)alamin Methyltransferase fromMethanosarcina acetivorans

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
He Fu ◽  
Michelle N. Goettge ◽  
William W. Metcalf
Keyword(s):  
Transfer Reaction ◽  
Biochemical Characterization ◽  
Detailed Knowledge ◽  
Coenzyme M ◽  
Metabolic Intermediate ◽  
Content Type ◽  
High Substrate ◽  
Methyl Transfer ◽  
Substrate Concentrations

ABSTRACTMethanogenesis from methylated substrates is initiated by substrate-specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, theMethanosarcina acetivoransMtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate (MMPA):coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, withkcatof 0.315 s−1and apparentKmfor MPA of 12 μM. CoM did not serve as a methyl acceptor; thus, a second unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, was also demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine, and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were coexpressed. Biochemical characterization of this complex was not feasible, because the corrinoid cofactor of this complex was in the inactive Co(II) state and was not reactivated by incubation with strong reductants. The MtsF protein, composed of both corrinoid and methyltransferase domains, copurifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism.IMPORTANCEMethylmercaptopropionate (MMPA) is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady-state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in the methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate, methyl coenzyme M. Therefore, the additional enzymes required for methanogenic MMPA catabolism await discovery.

scholarly journals Characterization of a Salmonella sugar kinase essential for the utilization of fructose-asparagine

2017 ◽  
Vol 95 (2) ◽  
pp. 304-309 ◽  
Author(s):  
Pradip K. Biswas ◽  
Edward J. Behrman ◽  
Venkat Gopalan
Keyword(s):  
Amino Acid ◽  
Mouse Models ◽  
Biochemical Characterization ◽  
Intestinal Inflammation ◽  
Sequential Action ◽  
Carbon And Nitrogen ◽  
Naturally Occurring ◽  
The Common ◽  
Sugar Kinase

Salmonella can utilize fructose-asparagine (F-Asn), a naturally occurring Amadori product, as its sole carbon and nitrogen source. Conversion of F-Asn to the common intermediates glucose-6-phosphate, aspartate, and ammonia was predicted to involve the sequential action of an asparaginase, a kinase, and a deglycase. Mutants lacking the deglycase are highly attenuated in mouse models of intestinal inflammation owing to the toxic build-up of the deglycase substrate. The limited distribution of this metabolic pathway in the animal gut microbiome raises the prospects for antibacterial discovery. We report the biochemical characterization of the kinase that was expected to transform fructose-aspartate to 6-phosphofructose-aspartate during F-Asn utilization. In addition to confirming its anticipated function, we determined through studies of fructose-aspartate analogues that this kinase exhibits a substrate-specificity with greater tolerance to changes to the amino acid (including the d-isomer of aspartate) than to the sugar.

Identification and Characterization of a Novel Carboxylesterase EstQ7 from a Soil Metagenomic Library

2021 ◽  
Author(s):  
Zhenzhen Yan ◽  
Liping Ding ◽  
Dandan Zou ◽  
Luyao Wang ◽  
Yuzhi Tan ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Metagenomic Library ◽  
Catalytic Triad ◽  
Recombinant Enzyme ◽  
Fatty Acid Esters ◽  
Dimensional Structure ◽  
Sequence Alignments ◽  
Acyltransferase Activity ◽  
Lipolytic Enzymes

Abstract A novel lipolytic gene, estq7, was identified from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42 kDa. Multiple sequence alignments showed that EstQ7 contained a pentapeptide motif GHSMG, and a putative catalytic triad Ser174–Asp306–His344. Interestingly, EstQ7 was found to have very little similarity to the characterized lipolytic enzymes. Phylogenetic analysis revealed that EstQ7 may be a member of a novel family of lipolytic enzymes. Biochemical characterization of the recombinant enzyme revealed that it constitutes a slightly alkalophilic, moderate thermophilic and highly active carboxylesterase against short chain fatty acid esters with optimum temperature 50 ℃ and pH 8.2. The Km and kcat values toward p-nitrophenyl acetate were determined to be 0.17 mM and 1910 S-1, respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Homology modeling of the three-dimensional structure of this new enzyme showed that it exhibits a typical α/β hydrolase fold, and the catalytic triad residues are spatially close. Molecular docking reveled the interactions between the enzyme and the ligand. The high levels of lipolytic activity of EstQ7, combined with its moderate thermophilic property, and acyltransferase activity, render this novel enzyme a promising candidate biocatalyst for food, pharmaceutical and biotechnological applications.

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