scholarly journals Thioglycoligase derived from fungal GH3 β-xylosidase is a multi-glycoligase with broad acceptor tolerance

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Manuel Nieto-Domínguez ◽  
Beatriz Fernández de Toro ◽  
Laura I. de Eugenio ◽  
Andrés G. Santana ◽  
Lara Bejarano-Muñoz ◽  
...  
Keyword(s):  
Active Site ◽  
Mutant Enzyme ◽  
Target Compound ◽  
Major Goal ◽  
Sugar Esters ◽  
Main Factor

Abstract The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal β-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the pKa of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis.

Synthesis of plantamajoside, a bioactive dihydroxyphenylethyl glycoside from Plantago major L.

Holzforschung ◽  
2006 ◽  
Vol 60 (5) ◽  
pp. 492-497 ◽  
Author(s):  
Toshinari Kawada ◽  
Yuko Yoneda ◽  
Ryuji Asano ◽  
Ippei Kan-no ◽  
Walther Schmid
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Total Synthesis ◽  
Caffeic Acid ◽  
Spectral Data ◽  
Natural Compound ◽  
Plantago Major ◽  
Target Compound ◽  
Sugar Esters ◽  
Nuclear Magnetic

Abstract The first total synthesis of plantamajoside (1), 2-(3′,4′-dihydroxylphenyl)ethyl-4-O-caffeoyl-3-O-(β-D-glucopyranosyl)-β-D-glucopyranoside, which is one of the dihydroxyphenylethyl glycosides (caffeic acid sugar esters), is described. Key intermediate 2, 2-[3′,4′-bis(O-benzyl)phenyl]ethyl 2,6-di-O-acetyl-4-O-[3′,4′-bis(O-benzyl)caffeoyl]-β-D-glucopyranoside was glycosylated with trichloroacetoimidoyl 2,3,4,6-tetra-O-acetyl-α-D-glycopyranoside (3) to afford plantamajoside derivative 4a, 2-[3′,4′-bis(O-benzyl)phenyl]ethyl 2,6-di-O-acetyl-4-O-[3′,4′-bis(O-benzyl)caffeoyl]-3-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-β-D-glucopyranoside, in 39% yield. Plantamajoside derivative 4a was successfully converted into the target compound, plantamajoside (1), through a series of de-protective procedures. 1H- and 13C nuclear magnetic resonance (NMR) spectral data of the synthesized plantamajoside (1) were identical to those of the natural compound.

scholarly journals Disruption of the crossover helix impairs dihydrofolate reductase activity in the bifunctional enzyme TS–DHFR from Cryptosporidium hominis

2009 ◽  
Vol 417 (3) ◽  
pp. 757-764 ◽  
Author(s):  
Melissa A. Vargo ◽  
W. Edward Martucci ◽  
Karen S. Anderson
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Reductase Activity ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Bifunctional Enzyme ◽  
Cryptosporidium Hominis ◽  
Single Polypeptide Chain ◽  
Species Specific

In contrast with most species, including humans, which have monofunctional forms of the folate biosynthetic enzymes TS (thymidylate synthase) and DHFR (dihydrofolate reductase), several pathogenic protozoal parasites, including Cryptosporidium hominis, contain a bifunctional form of the enzymes on a single polypeptide chain having both catalytic activities. The crystal structure of the bifunctional enzyme TS–DHFR C. hominis reveals a dimer with a ‘crossover helix’, a swap domain between DHFR domains, unique in that this helical region from one monomer makes extensive contacts with the DHFR active site of the other monomer. In the present study, we used site-directed mutagenesis to probe the role of this crossover helix in DHFR catalysis. Mutations were made to the crossover helix: an ‘alanine-face’ enzyme in which the residues on the face of the helix close to the DHFR active site of the other subunit were mutated to alanine, a ‘glycine-face’ enzyme in which the same residues were mutated to glycine, and an ‘all-alanine’ helix in which all residues of the helix were mutated to alanine. These mutant enzymes were studied using a rapid transient kinetic approach. The mutations caused a dramatic decrease in the DHFR activity. The DHFR catalytic activity of the alanine-face mutant enzyme was 30 s−1, the glycine-face mutant enzyme was 17 s−1, and the all-alanine helix enzyme was 16 s−1, all substantially impaired from the wild-type DHFR activity of 152 s−1. It is clear that loss of helix interactions results in a marked decrease in DHFR activity, supporting a role for this swap domain in DHFR catalysis. The crossover helix provides a unique structural feature of C. hominis bifunctional TS–DHFR that could be exploited as a target for species-specific non-active site inhibitors.

scholarly journals Salicortin: a repeat-attack new-mechanism-based Agrobacterium faecalis β-glucosidase inhibitor

1998 ◽  
Vol 332 (2) ◽  
pp. 367-371 ◽  
Author(s):  
Jianjun ZHU ◽  
Stephen G. WITHERS ◽  
Paul B. REICHARDT ◽  
Edward TREADWELL ◽  
Thomas P. CLAUSEN
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Mutant Enzyme ◽  
Quinone Methide ◽  
Free Solution ◽  
Tryptic Digest ◽  
Glucosidase Inhibitor ◽  
Inactive Form ◽  
Competitive Inhibitors

Salicortin, a natural product abundant in most members of the Salicaceae family, is a mechanism-based inactivator of Agrobacterium faecalis β-glucosidase. Inactivation is delayed in the presence of competitive inhibitors, thereby demonstrating the requirement for an enzyme-bound salicortin before inactivation. Product studies suggest that inactivation proceeds via a quinone methide intermediate formed by the fragmentation of the aglycone of salicortin while it is bound to the enzyme. Tryptic digest and HPLC/MS studies confirm the role of quinone methide attack and also show that the enzyme undergoes multiple modifications. In addition, when the inactivation was run in the presence of a mutant inactive form of the enzyme, HPLC/MS analyses clearly showed no modification of the mutant enzyme, demonstrating that the quinone methide does not exist in free solution and suggesting that inactivation is active-site directed.

scholarly journals Dipeptidyl Aminopeptidase IV from Stenotrophomonas maltophilia Exhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue

2008 ◽  
Vol 190 (23) ◽  
pp. 7819-7829 ◽  
Author(s):  
Yoshitaka Nakajima ◽  
Kiyoshi Ito ◽  
Tsubasa Toshima ◽  
Takashi Egawa ◽  
Heng Zheng ◽  
...  
Keyword(s):  
Active Site ◽  
Stenotrophomonas Maltophilia ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Subunit Structure ◽  
Hydrophobic Pocket ◽  
Cell Parameters ◽  
Replacement Method ◽  
Major Factors ◽  
Dipeptidyl Aminopeptidase

ABSTRACT The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-Å resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P43212, with unit cell parameters a = b = 105.9 Å and c = 161.9 Å. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal β-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.

scholarly journals Kinetic and stereochemical comparison of wild-type and active-site K145Q mutant enzyme of bacterial D-amino acid transaminase.

1993 ◽  
Vol 268 (10) ◽  
pp. 6932-6938
Author(s):  
M.B. Bhatia ◽  
S. Futaki ◽  
H. Ueno ◽  
J.M. Manning ◽  
D. Ringe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Mutant Enzyme ◽  
Wild Type

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 Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid

1991 ◽  
Vol 280 (2) ◽  
pp. 521-526 ◽  
Author(s):  
W J Man ◽  
Y Li ◽  
C D O'Connor ◽  
D C Wilton
Keyword(s):  
Catalytic Activity ◽  
Aspartic Acid ◽  
Active Site ◽  
Citrate Synthase ◽  
Condensation Reaction ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Reverse Direction ◽  
Acetyl Coa ◽  
Aspartic Acid Residue

The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp→Asn and Asp→Gly mutations. The Asp→Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563].

Studies on a Tyr residue critical for the binding of coenzyme and substrate in mouse 3(17)α-hydroxysteroid dehydrogenase (AKR1C21): structure of the Y224D mutant enzyme

2010 ◽  
Vol 66 (2) ◽  
pp. 198-204
Author(s):  
Urmi Dhagat ◽  
Satoshi Endo ◽  
Hiroaki Mamiya ◽  
Akira Hara ◽  
Ossama El-Kabbani
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Unique Feature ◽  
Hydroxysteroid Dehydrogenase ◽  
Kinetic Constants ◽  
Mutant Enzyme ◽  
Side Chains ◽  
Wild Type ◽  
Stereospecific Reduction ◽  
Flexible Loop

Mouse 3(17)α-hydroxysteroid dehydrogenase (AKR1C21) is the only aldo–keto reductase that catalyzes the stereospecific reduction of 3- and 17-ketosteroids to the corresponding 3(17)α-hydroxysteroids. The Y224D mutation of AKR1C21 reduced theKmvalue for NADP(H) by up to 80-fold and completely reversed the 17α stereospecificity of the enzyme. The crystal structure of the Y224D mutant at 2.3 Å resolution revealed that the mutation resulted in a change in the conformation of the flexible loop B, including the V-shaped groove, which is a unique feature of the active-site architecture of wild-type AKR1C21 and is formed by the side chains of Tyr224 and Trp227. Furthermore, mutations (Y224F and Q222N) of residues involved in forming the safety belt for binding of the coenzyme showed similar alterations in kinetic constants for 3α-hydroxy/3-ketosteroids and 17-hydroxy/ketosteroids compared with the wild type.

scholarly journals Re-design of Saccharomyces cerevisiae flavocytochrome b2: introduction of l-mandelate dehydrogenase activity

1998 ◽  
Vol 333 (1) ◽  
pp. 117-120 ◽  
Author(s):  
Rhona SINCLAIR ◽  
Graeme A. REID ◽  
Stephen K. CHAPMAN
Keyword(s):  
Lactate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Active Site ◽  
Mutant Enzyme ◽  
Activity Levels ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Activity Increase ◽  
Double Mutation ◽  
Flavocytochrome B2

Flavocytochrome b2 from Saccharomyces cerevisiaeis an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate. The double mutation of Ala-198 → Gly and Leu-230 → Ala results in an enzyme with a kcat value (25 °C) with l-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with l-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of l-mandelate at the active site of the enzyme. To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme. In addition, the double mutation of Ala-198 → Gly and Leu-230 → Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with l-mandelate than with l-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase.

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