QM/MM Studies Reveal How Substrate–Substrate and Enzyme–Substrate Interactions Modulate Retaining Glycosyltransferases Catalysis and Mechanism

Gómez, Hansel; Mendoza, Fernanda; Lluch, José M.; Masgrau, Laura

doi:10.1016/bs.apcsb.2015.06.004

Cited by 14 publications

(19 citation statements)

References 82 publications

(107 reference statements)

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“…Thus, WAT432 and 3-OH GLC seem to share a similar role in stabilizing the hydrolysis and transglycosylation TS, respectively, via interaction with the oxocarbenium ion. This kind of interactions have been identified in retaining glycosyltransferases to help the reaction, with the difference that due to the different disposition of the substrates they serve leaving group departure (Gómez et al, 2015). It is interesting to notice that the hydrogen atom of this 4-OH FUC group is in turn interacting with Glu392; the H4O FUC -OE1 GLU392 interaction becomes shorter along the hydrolysis and the T I paths (it is not present along the T II energy profile because a water molecule is located as a H-bonding bridge between O4 FUC and OE1 GLU392 ).…”

Section: Resultsmentioning

confidence: 99%

“…Reaction paths were scanned by performing harmonically restrained optimizations along a properly defined reaction coordinate for each chemical process in steps of 0.2 Å. Reaction coordinates were defined as linear combinations of the three main distances involved in each step, a definition that we have successfully used when modeling other carbohydrate-active enzymes (Gómez et al, 2015). Thus, RC = [ d (C1 FUC -O4D pNP )- d (C1 FUC -OE2 GLU338 )- d (H GLU164 -O4D pNP )] for glycosylation, RC = [ d (C1 FUC -OE2 GLU338 )- d (C1 FUC -O WAT431 )- d (H1 WAT431 -OE2 GLU164 )] for hydrolysis and RC = [ d (C1 FUC -OE2 GLU338 )- d (C1 FUC -O4 GLC )- d (H4O GLC -OE2 GLU164 )] for transglycosylation.…”

Section: Methodolgymentioning

confidence: 99%

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Comparing Hydrolysis and Transglycosylation Reactions Catalyzed by Thermus thermophilus β-Glycosidase. A Combined MD and QM/MM Study

et al. 2019

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The synthesis of oligosaccharides and other carbohydrate derivatives is of relevance for the advancement of glycosciences both at the fundamental and applied level. For many years, glycosyl hydrolases (GHs) have been explored to catalyze the synthesis of glycosidic bonds. In particular, retaining GHs can catalyze a transglycosylation (T) reaction that competes with hydrolysis (H). This has been done either employing controlled conditions in wild type GHs or by engineering new mutants. The goal, which is to increase the T/H ratio, has been achieved with moderate success in several cases despite the fact that the molecular basis for T/H modulation are unclear. Here we have used QM(DFT)/MM calculations to compare the glycosylation, hydrolysis and transglycosylation steps catalyzed by wild type Thermus thermophilus β-glycosidase (family GH1), a retaining glycosyl hydrolase for which a transglycosylation yield of 36% has been determined experimentally. The three transition states have a strong oxocarbenium character and ring conformations between 4 H 3 and 4 E. The atomic charges at the transition states for hydrolysis and transglycosylation are very similar, except for the more negative charge of the oxygen atom of water when compared to that of the acceptor Glc. The glycosylation transition state has a stronger S N 2 character than the deglycosylation ones and the proton transfer is less advanced. At the QM(PBE0/TZVP)/MM level, the TS for transglycosylation has shorter O4 GLC -C1 FUC (forming bond) distance and longer OE2 GLU338 -C1 FUC (breaking) distance than the hydrolysis one, although the H ACC proton is closer to the Glu164 base in the hydrolysis TS. The QM(SCC-DFTB)/MM free energy maxima show the inverted situation, although the hydrolysis TS presents significant structural fluctuations. The 3-OH GLC group of the acceptor Glc (transglycosylation) and WAT432 (neighbor water in hydrolysis) are identified to stabilize the oxocarbenium transition states through interaction with O5 FUC and O4 FUC . The analysis of interaction suggests that perturbing the Glu392-Fuc interaction could increase the T/H ratio, either by direct mutation of this residue or indirectly as reported experimentally in the Asn390I and Phe401S cases. The molecular understanding of similarities and differences between hydrolysis and transglycosylation steps may be of help in the design of new biocatalysts for glycan synthesis.

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Section: Resultsmentioning

confidence: 99%

Section: Methodolgymentioning

confidence: 99%

Comparing Hydrolysis and Transglycosylation Reactions Catalyzed by Thermus thermophilus β-Glycosidase. A Combined MD and QM/MM Study

et al. 2019

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“…Thus, positioning of the acceptor hydroxyl group relative to the donor anomeric carbon is critical in establishing the enzyme catalytic mechanism. These mechanisms have been reviewed in more detail elsewhere 25,[27][28][29][30] .…”

Section: Mechanistic Strategiesmentioning

confidence: 99%

Emerging structural insights into glycosyltransferase-mediated synthesis of glycans

2019

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Glycans linked to proteins and lipids play key roles in biology; thus, accurate replication of cellular glycans is crucial for maintaining function following cell division. The fact that glycans are not copied from genomic templates suggests that fidelity is provided by the catalytic templates of glycosyltransferases that accurately add sugars to specific locations on growing oligosaccharides. To form new glycosidic bonds, glycosyltransferases bind acceptor substrates and orient a specific hydroxyl group, frequently one of many, for attack of the donor sugar anomeric carbon. Several recent crystal structures of glycosyltransferases with bound acceptor substrates reveal that these enzymes have common core structures that function as scaffolds upon which variable loops are inserted to confer substrate specificity and correctly orient the nucleophilic hydroxyl group. The varied approaches for acceptor binding site assembly suggest an ongoing evolution of these loop regions provides templates for assembly of the diverse glycan structures observed in biology.

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“…With the increased concentration of the hydronium ions (Figure ), the glycosidic ether bond-breaking rate increased as well indicating that the rate of the QC–O–CQ (Q = glucose units) bond cleavage is proportional to the hydronium ion concentration. Nucleophiles such as the deprotonated amino acids can interact as stabilizers of the highly active oxocarbenium ion. , The charge stabilization capability of the corresponding amino acid nucleophile was proved via selective changes of the active site nucleophile, in Agrobacterium β-glucosidase . − The replacement of the active site nucleophile Glu358 (glutamic acid at 358 position of the enzyme peptide chain) to Asp caused 2500-fold rate reduction, corresponding to ∼19 kJ mol –1 destabilization. − The complete elimination of the charge (changing the Glu358 to Gln, Gln = glutamine) resulted in a 10 5 -fold rate reduction, corresponding to a destabilization by ∼36 kJ mol –1 . − …”

Section: Resultsmentioning

confidence: 99%

Catalytic Decomposition of the Oleaginous Yeast Cutaneotrichosporon Oleaginosus and Subsequent Biocatalytic Conversion of Liberated Free Fatty Acids

Braun

Lorenzen

Masri

et al. 2019

ACS Sustainable Chem. Eng.

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A single step catalytic cell wall lysis and triglyceride hydrolysis combined with the enzymatic conversion of lipids using the oleaginous yeast Cutaneotrichosporon oleaginosus (ATCC 20509) as a model is described. Catalytic decomposition of yeast cells resulted in hydrolysis of about a third of cellular polysaccharides and all triglycerides. Enzymatic processing of the lipid fraction with an oleate hydratase from Stenotrophomonas maltophilia led to conversion of oleic acid to 10-hydroxystearic acid (10-HSA) (50%) without additional purification. Cell wall polysaccharides were depolymerized by in situ formed amino acids from cell protein fragments. The activity of the in situ generated, free amino acids was higher compared to that of additionally added acids. Studies with the cellobiose and β-(1→3)-glucan indicated that glutamic and aspartic acids, which are the dominant amino acids in yeast cells, are surprisingly more effective in hydrolysis in aqueous phase than sulfuric acid. This points to a concerted mechanism of glycosidic ether bond cleavage catalyzed by amino acids rather than to a pathway catalyzed by hydronium ions. The overall yield of the presented downstream process at 453 K resulted in the release of 80% of total lipids.

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QM/MM Studies Reveal How Substrate–Substrate and Enzyme–Substrate Interactions Modulate Retaining Glycosyltransferases Catalysis and Mechanism

Cited by 14 publications

References 82 publications

Comparing Hydrolysis and Transglycosylation Reactions Catalyzed by Thermus thermophilus β-Glycosidase. A Combined MD and QM/MM Study

Comparing Hydrolysis and Transglycosylation Reactions Catalyzed by Thermus thermophilus β-Glycosidase. A Combined MD and QM/MM Study

Emerging structural insights into glycosyltransferase-mediated synthesis of glycans

Catalytic Decomposition of the Oleaginous Yeast Cutaneotrichosporon Oleaginosus and Subsequent Biocatalytic Conversion of Liberated Free Fatty Acids

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