Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD

AbstractTo adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.

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The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19800427 ◽

1980 ◽

Vol 45 (2) ◽

pp. 427-434 ◽

Cited By ~ 4

Author(s):

Kveta Heinrichová ◽

Rudolf Kohn

Keyword(s):

Acetyl Derivative ◽

Competitive Inhibitor ◽

Hydroxyl Groups ◽

Pectic Acid ◽

Substrate Complex ◽

Enzyme Substrate ◽

The Individual ◽

Degradation Degree ◽

Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

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Structures of β-glycosidase LXYL-P1-2 reveals the product binding state of GH3 family and a specific pocket for Taxol recognition

Communications Biology ◽

10.1038/s42003-019-0744-4 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Lin Yang ◽

Tian-Jiao Chen ◽

Fen Wang ◽

Li Li ◽

Wen-Bo Yu ◽

...

Keyword(s):

Crystal Structure ◽

Drug Delivery ◽

Industrial Production ◽

Binding Pocket ◽

Structural Basis ◽

Carrier Proteins ◽

Common Features ◽

Substrate Complex ◽

Enzyme Substrate ◽

Sugar Conformation

AbstractLXYL-P1-2 is one of the few xylosidases that efficiently catalyze the reaction from 7-β-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT), and is a potential enzyme used in Taxol industrial production. Here we report the crystal structure of LXYL-P1-2 and its XDT binding complex. These structures reveal an enzyme/product complex with the sugar conformation different from the enzyme/substrate complex reported previously in GH3 enzymes, even in the whole glycohydrolases family. In addition, the DT binding pocket is identified as the structural basis for the substrate specificity. Further structure analysis reveals common features in LXYL-P1-2 and Taxol binding protein tubulin, which might provide useful information for designing new Taxol carrier proteins for drug delivery.

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Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillus fumigatus

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013742 ◽

2020 ◽

Vol 295 (45) ◽

pp. 15407-15417

Author(s):

Daisuke Hira ◽

Takuya Onoue ◽

Takuji Oka

Keyword(s):

Aspergillus Fumigatus ◽

Structural Model ◽

Catalytic Domain ◽

Complex Structure ◽

Single Amino Acid ◽

Structural Basis ◽

Acceptor Substrate ◽

Substrate Complex ◽

The Core ◽

Enzyme Substrate

Fungal cell walls and their biosynthetic enzymes are potential targets for novel antifungal agents. Recently, two mannosyltransferases, namely core-mannan synthases A (CmsA/Ktr4) and B (CmsB/Ktr7), were found to play roles in the core-mannan biosynthesis of fungal-type galactomannan. CmsA/Ktr4 is an α-(1→2)-mannosyltransferase responsible for α-(1→2)-mannan biosynthesis in fungal-type galactomannan, which covers the cell surface of Aspergillus fumigatus. Strains with disrupted cmsA/ktr4 have been shown to exhibit strongly suppressed hyphal elongation and conidiation alongside reduced virulence in a mouse model of invasive aspergillosis, indicating that CmsA/Ktr4 is a potential novel antifungal candidate. In this study we present the 3D structures of the soluble catalytic domain of CmsA/Ktr4, as determined by X-ray crystallography at a resolution of 1.95 Å, as well as the enzyme and Mn2+/GDP complex to 1.90 Å resolution. The CmsA/Ktr4 protein not only contains a highly conserved binding pocket for the donor substrate, GDP-mannose, but also has a unique broad cleft structure formed by its N- and C-terminal regions and is expected to recognize the acceptor substrate, a mannan chain. Based on these crystal structures, we also present a 3D structural model of the enzyme–substrate complex generated using docking and molecular dynamics simulations with α-Man-(1→6)-α-Man-(1→2)-α-Man-OMe as the model structure for the acceptor substrate. This predicted enzyme–substrate complex structure is also supported by findings from single amino acid substitution CmsA/Ktr4 mutants expressed in ΔcmsA/ktr4 A. fumigatus cells. Taken together, these results provide basic information for developing specific α-mannan biosynthesis inhibitors for use as pharmaceuticals and/or pesticides.

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Structural insights into the substrate-binding mechanism for a novel chitosanase

Biochemical Journal ◽

10.1042/bj20140159 ◽

2014 ◽

Vol 461 (2) ◽

pp. 335-345 ◽

Cited By ~ 29

Author(s):

Qianqian Lyu ◽

Song Wang ◽

Wenhua Xu ◽

Baoqin Han ◽

Wanshun Liu ◽

...

Keyword(s):

Thermal Stability ◽

Substrate Binding ◽

Complex Structure ◽

Structural Basis ◽

Binding Mechanism ◽

Substrate Complex ◽

Cleavage Specificity ◽

First Time ◽

Structural Insights

We present the first chitosanase–substrate complex structure, and, in combination with a mutagenesis and thermal stability assay, we elucidate the chitosanase–substrate binding mechanism precisely for the first time. The structural basis for chitosanase cleavage specificity is analysed as well.

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Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

Microbiology Insights ◽

10.1177/11786361211024637 ◽

2021 ◽

Vol 14 ◽

pp. 117863612110246

Author(s):

Cheuk Yin Lai ◽

Ka Lun Ng ◽

Hao Wang ◽

Chui Chi Lam ◽

Wan Keung Raymond Wong

Keyword(s):

Escherichia Coli ◽

Cellulolytic Bacterium ◽

Systematic Screening ◽

Cellulomonas Fimi ◽

E Coli ◽

Substrate Complex ◽

Enzyme Substrate ◽

Glycosylation Sites ◽

Endogenous Proteins ◽

Cellulose Binding

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

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Conformational Analysis of the Enzyme-Substrate Complex in the Dehydrogenation of Sterols by Tetrahymena pyriformis

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)62451-2 ◽

1971 ◽

Vol 246 (3) ◽

pp. 561-568 ◽

Cited By ~ 4

Author(s):

William R. Nes ◽

P.A. Govinda Malya ◽

Frank B. Mallory ◽

Karen A. Ferguson ◽

Josephine R. Landrey ◽

...

Keyword(s):

Conformational Analysis ◽

Tetrahymena Pyriformis ◽

Substrate Complex ◽

Enzyme Substrate

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N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

Nucleic Acids Research ◽

10.1093/nar/gkab033 ◽

2021 ◽

Vol 49 (5) ◽

pp. 2684-2699

Author(s):

Ka-Weng Ieong ◽

Gabriele Indrisiunaite ◽

Arjun Prabhakar ◽

Joseph D Puglisi ◽

Måns Ehrenberg

Keyword(s):

Single Molecule ◽

Stop Codon ◽

Product Formation ◽

Release Factors ◽

Substrate Complex ◽

Peptidyl Transfer ◽

Enzyme Substrate ◽

Peptide Release ◽

Accuracy Ratio ◽

Codon Reading

Abstract We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

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Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

Canadian Journal of Biochemistry ◽

10.1139/o75-102 ◽

1975 ◽

Vol 53 (7) ◽

pp. 747-757 ◽

Cited By ~ 3

Author(s):

Graham J. Moore ◽

N. Leo Benoiton

Keyword(s):

Active Site ◽

Active Sites ◽

Kinetic Behavior ◽

Carboxypeptidase A ◽

Phenyllactic Acid ◽

Carboxypeptidase B ◽

Substrate Complex ◽

Enzyme Substrate ◽

Basic Peptides ◽

Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

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Structural insights into the substrate specificity of a glycoside hydrolase family 5 lichenase from Caldicellulosiruptor sp. F32

Biochemical Journal ◽

10.1042/bcj20170328 ◽

2017 ◽

Vol 474 (20) ◽

pp. 3373-3389 ◽

Cited By ~ 9

Author(s):

Dong-Dong Meng ◽

Xi Liu ◽

Sheng Dong ◽

Ye-Fei Wang ◽

Xiao-Qing Ma ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Glycoside Hydrolase ◽

Complex Structure ◽

Dynamics Simulation ◽

Structural Basis ◽

Glycoside Hydrolase Family ◽

Substrate Selectivity ◽

Glucose Residue ◽

Structural Insights

Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and a lichenan-degrading enzyme, and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, which is completely different from extensively studied GH16 lichenase that catalyses strict endo-hydrolysis of the β-1,4-glycosidic linkage adjacent to a 3-O-substitued glucose residue in the mixed-linked β-glucans. The crystal structure of F32EG5 was determined to 2.8 Å resolution, and the crystal structure of the complex of F32EG5 E193Q mutant and cellotetraose was determined to 1.7 Å resolution, which revealed that the exit subsites of substrate-binding sites contribute to both thermostability and substrate specificity of F32EG5. The sugar chain showed a sharp bend in the complex structure, suggesting that a substrate cleft fitting to the bent sugar chains in lichenan is a common feature of GH5 lichenases. The mechanism of thermostability and substrate selectivity of F32EG5 was further demonstrated by molecular dynamics simulation and site-directed mutagenesis. These results provide biochemical and structural insights into thermostability and substrate selectivity of GH5 lichenases, which have potential in industrial processes.

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