scholarly journals The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 599 ◽  
Author(s):  
Pavla Bojarová ◽  
Natalia Kulik ◽  
Michaela Hovorková ◽  
Kristýna Slámová ◽  
Helena Pelantová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reaction Medium ◽  
Hydrolytic Activity ◽  
High Yield ◽  
Amino Acid Residues ◽  
Yield Production ◽  
Selective Ligand ◽  
Selective Ligands ◽  
Galectin 3 ◽  
Fine Tune

N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by β-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. β-N-Acetylhexosaminidases exhibit activity towards both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of β-N-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the β-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcβ4GlcNAc disaccharide, a selective ligand of galectin-3.

scholarly journals Structural Insights into the Subclass B3 Metallo-β-Lactamase SMB-1 and the Mode of Inhibition by the Common Metallo-β-Lactamase Inhibitor Mercaptoacetate

2012 ◽  
Vol 57 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Jun-ichi Wachino ◽  
Yoshihiro Yamaguchi ◽  
Shigetarou Mori ◽  
Hiromasa Kurosaki ◽  
Yoshichika Arakawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Hydrolytic Activity ◽  
The Other ◽  
Amino Acid Residues ◽  
Hydrogen Bond Interaction ◽  
Content Type ◽  
Carboxylate Oxygen ◽  
Wide Range ◽  
Environmental Bacteria

ABSTRACTA novel subclass B3 metallo-β-lactamase (MBL), SMB-1, recently identified from aSerratia marcescensclinical isolate, showed a higher hydrolytic activity against a wide range of β-lactams than did the other subclass B3 MBLs, i.e., BJP-1 and FEZ-1, from environmental bacteria. To identify the mechanism underlying the differences in substrate specificity among the subclass B3 MBLs, we determined the structure of SMB-1, using 1.6-Å diffraction data. Consequently, we found that SMB-1 reserves a space in the active site to accommodate β-lactam, even with a bulky R1 side chain, due to a loss of amino acid residues corresponding to F31 and L226 of BJP-1, which protrude into the active site to prevent β-lactam from binding. The protein also possesses a unique amino acid residue, Q157, which probably plays a role in recognition of β-lactams via the hydrogen bond interaction, which is missing in BJP-1 and FEZ-1, whoseKmvalues for β-lactams are particularly high. In addition, we determined the mercaptoacetate (MCR)-complexed SMB-1 structure and revealed the mode of its inhibition by MCR: the thiolate group bridges to two zinc ions (Zn1 and Zn2). One of the carboxylate oxygen atoms of MCR makes contact with Zn2 and Ser221, and the other makes contact with T223 and a water molecule. Our results demonstrate the possibility that MCR could be a potent inhibitor for subclass B3 MBLs and that the screening technique using MCR as an inhibitor would be effective for detecting subclass B3 MBL producers.

scholarly journals A Novel Highly Thermostable Multifunctional Beta-Glycosidase from CrenarchaeonAcidilobus saccharovorans

Archaea ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Vadim M. Gumerov ◽  
Andrey L. Rakitin ◽  
Andrey V. Mardanov ◽  
Nikolai V. Ravin
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Half Life ◽  
Sequence Similarity ◽  
Hydrolytic Activity ◽  
Maximum Activity ◽  
Glycoside Hydrolases ◽  
Amino Acid Residues ◽  
High Tolerance ◽  
High Sequence Similarity

We expressed a putativeβ-galactosidase Asac_1390 from hyperthermophilic crenarchaeonAcidilobus saccharovoransinEscherichia coliand purified the recombinant enzyme. Asac_1390 is composed of 490 amino acid residues and showed high sequence similarity to family 1 glycoside hydrolases from various thermophilic Crenarchaeota. The maximum activity was observed at pH 6.0 and 93°C. The half-life of the enzyme at 90°C was about 7 hours. Asac_1390 displayed high tolerance to glucose and exhibits hydrolytic activity towards cellobiose and various aryl glucosides. The hydrolytic activity withp-nitrophenyl (pNP) substrates followed the order pNP-β-D-galactopyranoside (328 U mg−1), pNP-β-D-glucopyranoside (246 U mg−1), pNP-β-D-xylopyranoside (72 U mg−1), and pNP-β-D-mannopyranoside (28 U mg−1). Thus the enzyme was actually a multifunctionalβ-glycosidase. Therefore, the utilization of Asac_1390 may contribute to facilitating the efficient degradation of lignocellulosic biomass and help enhance bioconversion processes.

scholarly journals Crystal Structure of Mox-1, a Unique Plasmid-Mediated Class C β-Lactamase with Hydrolytic Activity towards Moxalactam

2014 ◽  
Vol 58 (7) ◽  
pp. 3914-3920 ◽  
Author(s):  
Takuma Oguri ◽  
Takamitsu Furuyama ◽  
Takashi Okuno ◽  
Yoshikazu Ishii ◽  
Kazuhiro Tateda ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hydrolytic Activity ◽  
Crystallographic Structure ◽  
Structural Flexibility ◽  
Amino Acid Residues ◽  
Structural Elements ◽  
X Ray ◽  
Binding Cavity ◽  
Class C ◽  
Unique Plasmid

ABSTRACTMox-1 is a unique plasmid-mediated class C β-lactamase that hydrolyzes penicillins, cephalothin, and the expanded-spectrum cephalosporins cefepime and moxalactam. In order to understand the unique substrate profile of this enzyme, we determined the X-ray crystallographic structure of Mox-1 β-lactamase at a 1.5-Å resolution. The overall structure of Mox-1 β-lactamase resembles that of other AmpC enzymes, with some notable exceptions. First, comparison with other enzymes whose structures have been solved reveals significant differences in the composition of amino acids that make up the hydrogen-bonding network and the position of structural elements in the substrate-binding cavity. Second, the main-chain electron density is not observed in two regions, one containing amino acid residues 214 to 216 positioned in the Ω loop and the other in the N terminus of the B3 β-strand corresponding to amino acid residues 303 to 306. The last two observations suggest that there is significant structural flexibility of these regions, a property which may impact the recognition and binding of substrates in Mox-1. These important differences allow us to propose that the binding of moxalactam in Mox-1 is facilitated by the avoidance of steric clashes, indicating that a substrate-induced conformational change underlies the basis of the hydrolytic profile of Mox-1 β-lactamase.

The 5-ketofructose reductase of Gluconobacter sp. strain CHM43 is a novel class in the shikimate dehydrogenase family

2021 ◽  
Author(s):  
Thuy Minh Nguyen ◽  
Masaru Goto ◽  
Shohei Noda ◽  
Minenosuke Matsutani ◽  
Yuki Hodoya ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Binding Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
High Yield ◽  
Strong Increase ◽  
Amino Acid Residues ◽  
Shikimate Dehydrogenase ◽  
Substrate Binding Site

Gluconobacter sp. CHM43 oxidizes mannitol to fructose and then does fructose to 5-keto-D-fructose (5KF) in the periplasmic space. Since NADPH-dependent 5KF reductase was found in the soluble fraction of Gluconobacter spp., 5KF might be transported into the cytoplasm and metabolized. Here we identified the GLF_2050 gene as the kfr gene encoding 5KF reductase (KFR). A mutant strain devoid of the kfr gene showed lower KFR activity and no 5KF consumption. The crystal structure revealed that KFR is similar to NADP + -dependent shikimate dehydrogenase (SDH), which catalyzes the reversible NADP + -dependent oxidation of shikimate to 3-dehydroshikimate. We found that several amino acid residues in the putative substrate-binding site of KFR were different from those of SDH. Phylogenetic analyses revealed that only a subclass in the SDH family containing KFR conserved such a unique substrate-binding site. We constructed KFR derivatives with amino acid substitutions, including replacement of Asn21 in the substrate-binding site with Ser that is found in SDH. The KFR-N21S derivative showed a strong increase in the K M value for 5KF, but a higher shikimate oxidation activity than wild-type KFR, suggesting that Asn21 is important for 5KF binding. In addition, the conserved catalytic dyad Lys72 and Asp108 were individually substituted for Asn. The K72N and D108N derivatives showed only negligible activities without a dramatic change in the K M value for 5KF, suggesting a similar catalytic mechanism to that of SDH. Taken together, we suggest that KFR is a new member of the SDH family. Importance A limited number of species of acetic acid bacteria, such as Gluconobacter sp. strain CHM43, produce 5-ketofructose at a high yield, a potential low calorie sweetener. Here we show that an NADPH-dependent 5-ketofructose reductase (KFR) is involved in 5-ketofructose degradation and we characterize this enzyme with respect to its structure, phylogeny, and function. The crystal structure of KFR was similar to that of shikimate dehydrogenase, which is functionally crucial in the shikimate pathway in bacteria and plants. Phylogenetic analysis suggested that KFR is positioned in a small sub-group of the shikimate dehydrogenase family. Catalytically important amino acid residues were also conserved and their relevance was experimentally validated. Thus, we propose KFR as a new member of shikimate dehydrogenase family.

scholarly journals Mutational properties of amino acid residues: implications for evolvability of phosphorylatable residues

2012 ◽  
Vol 367 (1602) ◽  
pp. 2584-2593 ◽  
Author(s):  
Pau Creixell ◽  
Erwin M. Schoof ◽  
Chris Soon Heng Tan ◽  
Rune Linding
Keyword(s):  
Amino Acid ◽  
Hot Spot ◽  
Phosphorylation Site ◽  
Chemical Properties ◽  
Implicit Assumption ◽  
Amino Acid Residues ◽  
Functional Protein ◽  
Ancestral Sequences ◽  
Physico Chemical ◽  
Fine Tune

As François Jacob pointed out over 30 years ago, evolution is a tinkering process, and, as such, relies on the genetic diversity produced by mutation subsequently shaped by Darwinian selection. However, there is one implicit assumption that is made when studying this tinkering process; it is typically assumed that all amino acid residues are equally likely to mutate or to result from a mutation. Here, by reconstructing ancestral sequences and computing mutational probabilities for all the amino acid residues, we refute this assumption and show extensive inequalities between different residues in terms of their mutational activity. Moreover, we highlight the importance of the genetic code and physico-chemical properties of the amino acid residues as likely causes of these inequalities and uncover serine as a mutational hot spot. Finally, we explore the consequences that these different mutational properties have on phosphorylation site evolution, showing that a higher degree of evolvability exists for phosphorylated threonine and, to a lesser extent, serine in comparison with tyrosine residues. As exemplified by the suppression of serine's mutational activity in phosphorylation sites, our results suggest that the cell can fine-tune the mutational activities of amino acid residues when they reside in functional protein regions.

Physicochemical Properties of Ferredoxin from Chlamydomonas reinhardii

1985 ◽  
Vol 40 (5-6) ◽  
pp. 373-378 ◽  
Author(s):  
Francisco Galván ◽  
Antonio J. Márquez ◽  
Emilio Fernández
Keyword(s):  
Amino Acid ◽  
Average Molecular Weight ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
High Yield ◽  
Amino Acid Residues ◽  
Chlamydomonas Reinhardii ◽  
Midpoint Potential ◽  
Stokes Radius ◽  
Fast Procedure

Abstract Ferredoxin from Chlamydomonas reinhardii has been purified to electrophoretic homogeneity by an easy and fast procedure with a high yield (25 -30 mg/250 g wet weight of cells). An average molecular weight of 11800 was calculated from sedimentation coefficient (1.70 S) and Stokes radius (1.75 nm) data, sodium dodecyl sulfate-electrophoresis, and amino acid composition. Absorption spectrum show ed maxima at 276, 330, 420 and 460 nm in the oxidized form, with an absorption ratio (A420/A276) of 0.54 and an extinction coefficient of 8.38 mᴍ-1· cm -1 at 420 nm. R educed ferredoxin show ed a single peak at 276 nm with shoulders at 284, 310, 390, 469 and 537 nm and at liquid helium temperatures gave EPR signals at g = 1.877, 1.951 and 2.045. The protein has an isoelectric point of 3.30, and one (2Fe-2S)-cluster per molecule with a midpoint potential, at pH 7.5, of -410 mV (n=1) . The m olecule of C. reinhardii ferredoxin consists of 95-99 amino acid residues which includes the full com plement of amino acids, being alanine the most abundant.

Sign in / Sign up

Export Citation Format

Share Document