scholarly journals Effect of Leu277 on Disproportionation and Hydrolysis Activity in Bacillus stearothermophilus NO2 Cyclodextrin Glucosyltransferase

2021 ◽  
Author(s):  
Demin Kong ◽  
Lei Wang ◽  
Lingqia Su ◽  
Jing Wu
Keyword(s):  
Bacillus Stearothermophilus ◽  
Structural Data ◽  
Side Chain ◽  
Future Research ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Multiple Sequence ◽  
Binding Cleft ◽  
Almost All ◽  
Hydrolysis Activity

The disproportionation activity of cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19) can be used to convert small molecules into glycosides, thereby enhancing their solubility and stability. However, CGTases also exhibit a competing hydrolysis activity. The +2 subsite of the substrate binding cleft plays an important role in both the disproportionation and hydrolysis activities, but almost all known mutations at this site decrease disproportionation activity. In this study, Leu277 of the CGTase from Bacillus stearothermophilus NO2, located near both the +2 subsite and the catalytic acid/base Glu253, was modified to assess the effect of side chain size at this position on disproportionation and hydrolysis activities. The best mutant, L277M, exhibited a reduced Km for the acceptor substrate maltose (0.48 mM versus 0.945 mM) and an increased kcat/Km (1175 s−1mM−1 versus 686.1 s−1mM−1), compared with those of the wild-type enzyme. The disproportionation to hydrolysis ratio of L277M was 2.4-fold greater than that of the wild-type. Existing structural data were combined with a multiple sequence alignment and Gly282 mutations to examine the mechanism behind the effects of the Leu277mutations. The Gly282 mutations were included to aid a molecular-dynamics (MD) analysis and the comparison of crystal structures. They reveal that changes to a hydrophobic cluster near Glu253 and the hydrophobicity of the +2 subsite combine to produce the observed effects. Importance In this study, mutations that enhance the disproportionation to hydrolysis ratio of a CGTase have been discovered. For example, the disproportionation to hydrolysis ratio of the L277M mutant of Bacillus stearothermophilus NO2 CGTase was 2.4-fold greater than that of the wild-type. The mechanism behind the effects of these mutations is explained. This paper opens up other avenues for future research into the disproportionation and hydrolysis activities of CGTases. Productive mutations are no longer limited to the acceptor subsite, since mutations that indirectly affect the acceptor subsite also enhance enzymatic activity.

Site-directed mutagenesis of farnesyl diphosphate synthase; effect of substitution on the three carboxyl-terminal amino acids

1994 ◽  
Vol 72 (1) ◽  
pp. 75-79 ◽  
Author(s):  
Tanetoshi Koyama ◽  
Kazuhiro Saito ◽  
Kyozo Ogura ◽  
Shusei Obata ◽  
Ayumi Takeshita
Keyword(s):  
Amino Acids ◽  
Bacillus Stearothermophilus ◽  
Farnesyl Diphosphate ◽  
Site Directed Mutagenesis ◽  
Farnesyl Diphosphate Synthase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Directed Mutation ◽  
Catalytic Function ◽  
Terminal Amino

Site-directed mutation was introduced into the gene for the farnesyl diphosphate synthase of Bacillus stearothermophilus. To investigate the significance of the three C-terminal amino acids, where arginine is completely conserved throughout the farnesyl diphosphate synthases of prokaryotes and eukaryotes, three kinds of mutant enzymes, R295V, D296G, and H297L, which have replacements of arginine-295 with valine, aspartate-296 with glycine, and histidine-297 with leucine, respectively, were overproduced and purified to homogeneity. All of the three mutant enzymes showed similar catalytic activities to that of the wild-type enzyme, indicating that the basic amino acids including the conserved arginine in the C-terminal region are not essential for catalytic function. They were also similar to the wild-type enzyme with respect to pH optima, thermostability, reaction product, and kinetic parameters for allylic substrates. However, their Km values for isopentenyl diphosphate are approximately twice that of the wild type.

scholarly journals Structure–activity correlations of variant forms of the B pentamer ofEscherichia colitype II heat-labile enterotoxin LT-IIb with Toll-like receptor 2 binding

2012 ◽  
Vol 68 (12) ◽  
pp. 1604-1612 ◽  
Author(s):  
Vivian Cody ◽  
Jim Pace ◽  
Hesham F. Nawar ◽  
Natalie King-Lyons ◽  
Shuang Liang ◽  
...  
Keyword(s):  
Structural Data ◽  
Binding Activity ◽  
Side Chain ◽  
Innate Immune Responses ◽  
Toll Like Receptor ◽  
Wild Type ◽  
B Subunit ◽  
Heat Labile ◽  
Pore Opening ◽  
Toll Like Receptor 2

The pentameric B subunit of the type II heat-labile enterotoxin ofEscherichia coli(LT-IIb-B5) is a potent signaling molecule capable of modulating innate immune responses. It has previously been shown that LT-IIb-B5, but not the LT-IIb-B5Ser74Asp variant [LT-IIb-B5(S74D)], activates Toll-like receptor (TLR2) signaling in macrophages. Consistent with this, the LT-IIb-B5(S74D) variant failed to bind TLR2, in contrast to LT-IIb-B5and the LT-IIb-B5Thr13Ile [LT-IIb-B5(T13I)] and LT-IIb-B5Ser74Ala [LT-IIb-B5(S74A)] variants, which displayed the highest binding activity to TLR2. Crystal structures of the Ser74Asp, Ser74Ala and Thr13Ile variants of LT-IIb-B5have been determined to 1.90, 1.40 and 1.90 Å resolution, respectively. The structural data for the Ser74Asp variant reveal that the carboxylate side chain points into the pore, thereby reducing the pore size compared with that of the wild-type or the Ser74Ala variant B pentamer. On the basis of these crystallographic data, the reduced TLR2-binding affinity of the LT-IIb-B5(S74D) variant may be the result of the pore of the pentamer being closed. On the other hand, the explanation for the enhanced TLR2-binding activity of the LT-IIb-B5(S74A) variant is more complex as its activity is greater than that of the wild-type B pentamer, which also has an open pore as the Ser74 side chain points away from the pore opening. Data for the LT-IIb-B5(T13I) variant show that four of the five variant side chains point to the outside surface of the pentamer and one residue points inside. These data are consistent with the lack of binding of the LT-IIb-B5(T13I) variant to GD1a ganglioside.

scholarly journals Rapid burst kinetics in the hydrolysis of 4-nitrophenyl acetate by penicillin G acylase from Kluyvera citrophila. Effects of mutation F360V on rate constants for acylation and de-acylation

1996 ◽  
Vol 316 (2) ◽  
pp. 409-412 ◽  
Author(s):  
A Roa ◽  
M L Goble ◽  
J L García ◽  
C Acebal ◽  
R Virden
Keyword(s):  
Rate Constants ◽  
Acetyl Derivative ◽  
Penicillin G Acylase ◽  
Penicillin G ◽  
Side Chain ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Burst Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1·s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1·s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.

scholarly journals A Biocatalytic Approach to a Key Intermediate for the Synthesis of the COVID-19 Experimental Drug Molnupiravir

2021 ◽  
Author(s):  
Ashleigh Burke ◽  
William Birmingham ◽  
Ying Zhuo ◽  
Bruna Zuculoto da Costa ◽  
Rebecca Crawshaw ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Active Site ◽  
Cytidine Deaminase ◽  
Good Activity ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Experimental Drug ◽  
Hydrolysis Activity

Herein we report the conversion of cytidine 2 to N-hydroxycytidine 7 catalysed by cytidine deaminase (CD). The wild-type enzyme operates efficiently at high sustrate loadings and hydroxylamine concentrations to favor N-hydroxy-cytidine formation over uridine. Although the wild-type enzyme demonstrated good activity, we were able to further enhance the ratio of N-hydroxycytidine to uridine produced through directed evolution of CD. In particular, a T123G mutation close to the active site dramatically reduces cytidine hydrolysis activity whilst preserving desired amination activty. The approach reported provides a new route to a key intermediate for the COVID-19 experimental drug Molnupiravir 1.

scholarly journals Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases

2013 ◽  
Vol 288 (20) ◽  
pp. 14087-14097 ◽  
Author(s):  
Zachary D. Nagel ◽  
Shujian Cun ◽  
Judith P. Klinman
Keyword(s):  
Long Range ◽  
Active Site ◽  
Bacillus Stearothermophilus ◽  
Isotope Effects ◽  
Thermostable Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Aromatic Side Chain ◽  
Alcohol Dehydrogenases ◽  
Rate Determining Step

A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 Å away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496–499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases.

scholarly journals The mutation Lys234His yields a class A β-lactamase with a novel pH-dependence

1991 ◽  
Vol 278 (3) ◽  
pp. 673-678 ◽  
Author(s):  
J Brannigan ◽  
A Matagne ◽  
F Jacob ◽  
C Damblon ◽  
B Joris ◽  
...  
Keyword(s):  
Active Site ◽  
Positive Charge ◽  
Ph Dependence ◽  
Side Chain ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Beta Lactamases ◽  
Class A ◽  
Transition State Stabilization

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

scholarly journals Contribution of a buried aspartate residue towards the catalytic efficiency and structural stability of Bacillus stearothermophilus lactate dehydrogenase

1994 ◽  
Vol 300 (2) ◽  
pp. 491-499 ◽  
Author(s):  
T J Nobbs ◽  
A Cortés ◽  
J L Gelpi ◽  
J J Holbrook ◽  
T Atkinson ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Lactate Dehydrogenase ◽  
Active Site ◽  
Bacillus Stearothermophilus ◽  
Mutant Enzyme ◽  
Hydroxyl Group ◽  
Side Chain ◽  
General Effect ◽  
Carboxylate Group ◽  
Wild Type

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

2021 ◽  
Vol 22 (6) ◽  
pp. 3230
Author(s):  
Mireia Castejón-Vilatersana ◽  
Magda Faijes ◽  
Antoni Planas
Keyword(s):  
Hydrolytic Activity ◽  
Bifidobacterium Bifidum ◽  
Glycoside Hydrolases ◽  
Type I ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Transglycosylation Activity ◽  
Binding Cleft ◽  
Breast Fed ◽  
Enzyme Variant

The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10-30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.

scholarly journals Selective modification by transglutaminase of a glutamine side chain in the hinge region of the histidine-388→glutamine mutant of yeast phosphoglycerate kinase

1991 ◽  
Vol 273 (1) ◽  
pp. 73-78 ◽  
Author(s):  
P J Coussons ◽  
S M Kelly ◽  
N C Price ◽  
C M Johnson ◽  
B Smith ◽  
...  
Keyword(s):  
Phosphoglycerate Kinase ◽  
Hinge Region ◽  
Mutant Enzyme ◽  
Side Chain ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Significant Change

The transglutaminase-catalysed incorporation of putrescine and monodansylcadaverine into yeast phosphoglycerate kinase has been studied. There is little incorporation of the amines into wild-type enzyme, but nearly stoichiometric incorporation into the histidine-388----glutamine mutant enzyme. C.d. studies show that the overall structure of the mutant enzyme is very similar to that of the wild-type enzyme. Incorporation of the amines into the mutant enzyme causes no significant change in its activity. Glutamine-388 was shown, by isolation and sequencing of the modified peptide, to be the site of incorporation of monodansylcadaverine into the mutant enzyme. The specificity of the transglutaminase reaction is discussed in the light of available data.

scholarly journals Electrostatic stabilization in a pre-organized polar active site: the catalytic role of Lys-80 in Candida tenuis xylose reductase (AKR2B5) probed by site-directed mutagenesis and functional complementation studies

2005 ◽  
Vol 389 (2) ◽  
pp. 507-515 ◽  
Author(s):  
Regina Kratzer ◽  
Bernd Nidetzky
Keyword(s):  
Carbonyl Group ◽  
Acid Catalysis ◽  
Xylose Reductase ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Linear Free Energy Relationship ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
General Acid ◽  
Group Reduction

Lys-80 of Candida tenuis xylose reductase (AKR2B5) is conserved throughout the aldo–keto reductase protein superfamily and may prime the nearby Tyr-51 for general acid catalysis to NAD(P)H-dependent carbonyl group reduction. We have examined the catalytic significance of side-chain substitutions in two AKR2B5 mutants, Lys-80→Ala (K80A) and Asp-46→Asn Lys-80→Ala (D46N K80A), using steady-state kinetic analysis and restoration of activity with external amines. Binding of NAD+ (Kd=24 μM) and NADP+ (Kd=0.03 μM) was 10- and 40-fold tighter in K80A than the wild-type enzyme, whereas binding of NADH (Kd=51 μM) and NADPH (Kd=19 μM) was weakened 2- and 16-fold in this mutant respectively. D46N K80A bound NAD(P)H and NAD(P)+ uniformly approx. 5-fold less tightly than the wild-type enzyme. The second-order rate constant for non-covalent restoration of NADH-dependent reductase activity (kmax/Kamine) by protonated ethylamine was 0.11 M−1·s−1 for K80A, whereas no detectable rescue occurred for D46N K80A. After correction for effects of side-chain hydrophobicity, we obtained a linear free energy relationship of log (kmax/Kamine) and amine group pKa (slope=+0.29; r2=0.93) at pH 7.0. pH profiles of log (kcat/Km) for carbonyl group reduction by wild-type and D46N K80A revealed identical and kinetically unperturbed pKa values of 8.50 (±0.20). Therefore the protonated side chain of Lys-80 is not an essential activator of general acid catalysis by AKR2B5. Stabilized structurally through the salt-link interaction with the negatively charged Asp-46, it is proposed to pull the side chain of Tyr-51 into the catalytic position, leading to a preorganized polar environment of overall neutral charge, in which approximation of uncharged reactive groups is favoured and thus hydride transfer from NAD(P)H is strongly preferred. Lys-80 affects further the directional preference of AKR2B5 for NAD(P)H-dependent reduction by increasing NAD(P)H compared with NAD(P)+-binding selectivity.

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