Enzyme–Substrate Complex Structures of a GH39 β-Xylosidase from Geobacillus stearothermophilus

Zearalenone hydrolase (ZHD) is an α/β-hydrolase that detoxifies and degrades the lactone zearalenone (ZEN), a naturally occurring oestrogenic mycotoxin that contaminates crops. Several apoenzyme and enzyme–substrate complex structures have been reported in the resolution range 2.4–2.6 Å. However, the properties and mechanism of this enzyme are not yet fully understood. Here, a 1.60 Å resolution structure of a ZHD–product complex is reported which was determined from a C-terminally His6-tagged ZHD crystal soaked with 2 mMZEN for 30 min. It shows that after the lactone-bond cleavage, the phenol-ring region moves closer to residues Leu132, Tyr187 and Pro188, while the lactone-ring region barely moves. Comparisons of the ZHD–substrate and ZHD–product structures show that the hydrophilic interactions change, especially Trp183 N∊1, which shifts from contacting O2 to O12′, suggesting that Trp183 is responsible for the unidirectional translational movement of the phenol ring. This structure provides information on the final stage of the catalytic mechanism of zearalenone hydrolysis.

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Enzyme–substrate complex structures of CYP154C5 shed light on its mode of highly selective steroid hydroxylation

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714019129 ◽

2014 ◽

Vol 70 (11) ◽

pp. 2875-2889 ◽

Cited By ~ 11

Author(s):

Konrad Herzog ◽

Paula Bracco ◽

Akira Onoda ◽

Takashi Hayashi ◽

Kurt Hoffmann ◽

...

Keyword(s):

Polar Regions ◽

Complex Structures ◽

P450 Monooxygenase ◽

Hormone Synthesis ◽

Substrate Complex ◽

Enzyme Substrate ◽

Structural Differences ◽

Key Enzyme ◽

Steroid Binding ◽

Shed Light

CYP154C5 fromNocardia farcinicais a bacterial cytochrome P450 monooxygenase active on steroid molecules. The enzyme has recently been shown to exhibit exclusive regioselectivity and stereoselectivity in the conversion of various pregnans and androstans, yielding 16α-hydroxylated steroid products. This makes the enzyme an attractive candidate for industrial application in steroid hormone synthesis. Here, crystal structures of CYP154C5 in complex with four different steroid molecules were solved at resolutions of up to 1.9 Å. These are the first reported P450 structures from the CYP154 family in complex with a substrate. The active site of CYP154C5 forms a flattened hydrophobic channel with two opposing polar regions, perfectly resembling the size and polarity distribution of the steroids and thus resulting in highly specific steroid binding withKdvalues in the range 10–100 nM. Key enzyme–substrate interactions were identified that accounted for the exclusive regioselectivity and stereoselectivity of the enzyme. Additionally, comparison of the four CYP154C5–steroid structures revealed distinct structural differences, explaining the observed variations in kinetic data obtained for this P450 with the steroids pregnenolone, dehydroepiandrosterone, progesterone, androstenedione, testosterone and nandrolone. This will facilitate the generation of variants with improved activity or altered selectivity in the future by means of protein engineering.

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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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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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Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on formation of the enzyme-substrate complex

Biochemistry ◽

10.1021/bi00315a008 ◽

1984 ◽

Vol 23 (20) ◽

pp. 4557-4564 ◽

Cited By ~ 57

Author(s):

Jan L. M. L. Van Rijn ◽

Jose W. P. Govers-Riemslag ◽

Robert F. A. Zwaal ◽

Jan Rosing

Keyword(s):

Kinetic Studies ◽

Activation Effect ◽

Substrate Complex ◽

Factor Va ◽

Enzyme Substrate ◽

Prothrombin Activation

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Kinetics of Bond Cleavages at Kallidin Release by Tissue Kallikrein: Cleavage of Two Peptide Bonds in a Single Enzyme-Substrate Complex ?

Recent Progress on Kinins ◽

10.1007/978-3-0348-7321-5_11 ◽

1992 ◽

pp. 82-88

Author(s):

F. Fiedler ◽

H. Hinz

Keyword(s):

Tissue Kallikrein ◽

Peptide Bonds ◽

Substrate Complex ◽

Enzyme Substrate ◽

Kinetics Of ◽

Single Enzyme

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Fragment-derived modulators of an industrial β-glucosidase

Biochemical Journal ◽

10.1042/bcj20200507 ◽

2020 ◽

Vol 477 (22) ◽

pp. 4383-4395

Author(s):

Eleni Makraki ◽

John F. Darby ◽

Marta G. Carneiro ◽

James D. Firth ◽

Alex Heyam ◽

...

Keyword(s):

Binding Site ◽

Glycoside Hydrolase ◽

Mixed Model ◽

Specific Binding ◽

Fold Increase ◽

Kinetic Assay ◽

Nmr Spectrum ◽

Substrate Complex ◽

Enzyme Substrate ◽

Maximum Activation

A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme–substrate complex.

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