Biocatalysts from Aplysia 30.2 Biocatalytic Methodologies to Access Glycosides and Oligosaccharides 733
Part F | 30.2
can alter or modulate biological functions. Enzymes responsible for the synthesis of glycosidic linkage have
been recognized as transglycosylases and named glycosyltransferases, specifying the glycosyl donor and the
reaction product. These enzymes transfer sugar moieties from the activated donor to specific acceptors,
forming a specific glycosidic bond, and are responsible
in vivo for the synthesis of most cell-surface glycoconjugates, using eight common sugar nucleotides as
activated donors (Leloir pathway). Sugar phosphates
can act as donors for other glycosyltransferases (nonLeloir pathway).
Glycoside hydrolases (glycosidases), represent another widespread group of enzymes involved in the
carbohydrate metabolism, being responsible for the hydrolysis of glycosidic linkages; these enzymes can act
as exo or endoglycosidases and are involved in a series
of biological events of important nature, such as energy
uptake in processes inherent in cell wall metabolism,
in glycan processing during in vivo glycoprotein synthesis, etc. Glycosidases and endoglycosidases are enzymes that are currently leading to new expectations in
research and development due not only to their natural
capability to act on the internal part of an oligosaccharide chain, but also to the possibility of transferring
a whole carbohydrate block to a new acceptor, thus enabling easy reconstruction of complex glycoconjugates
(e.g., glycopeptides, bioactive oligosaccharides, etc.).
In addition, the profitable use of the hydrolytic capabilities of glycosyl hydrolases for mass preparation of
oligosaccharides from natural marine or terrestrial polymers should be mentioned here [30.10].
The stereochemistry of the mechanisms of glycoside hydrolases, analyzed more than 50 years
ago [30.11], allowed the classification of glycoside hydrolases in inverting and retaining enzymes in accord
to the anomeric configuration found in the product with
respect to that in the starting substrate. Very recently,
it has become clear that other mechanisms are evolved,
such as the one based on elimination [30.12].
In the inverting glycoside hydrolases, the glycosidic
substrate, a water molecule, and two carboxylic acid
groups represent the key elements of the chemical machinery within the active site of the enzyme. The two
carboxyl groups acting as general acid and general base
are distant; between them there is a (10:5 Å) value double that of retaining enzymes. This creates room for
the substrate and a water molecule to access the active
site. Hydrolysis occurs via a single displacement mechanism that involves an oxocarbenium ion-like transition
state. The product has the opposite configuration at the
anomeric center with respect to the substrate. If an inverting enzyme catalyzes a transglycosylation reaction
transferring the sugar on an acceptor other than water, the product of this reaction would be the inverted
anomer of the starting glycoside. This transglycosylating activity is never found with these enzymes; in
fact, they would have to be anomerically indiscriminate
to possess such activity. However, inverting enzymes
catalyze oligosaccharide synthesis via reversal of the
hydrolysis reaction; in the presence of a free sugar and
alcohol acceptor, back reaction can produce a glycoside
(see below).
A reaction characterized by a double displacement
mechanism is operative in retaining glycoside hydrolases; two sequential steps can be distinguished in
this case. In the first, the substrate binds the active
site, then a nucleophilic group (aspartic acid, glutamic
acid, or histidine) attacks the anomeric carbon atom.
Protonation of aglycone oxygen by an acid proton,
with concomitant glycosidic bond cleavage and ester
bond formation, leads to the formation of the covalent ester glucosyl-enzyme intermediate, characterized
by anomeric inversion at C1. In the second step, a hydroxyl group from a water molecule (in the case of
the hydrolysis reaction) or from a different residue (for
transglycosylation reactions) attacks the ester bond of
the intermediate with re-inversion of configuration of
the anomeric carbon. As a result, the final product has
the same anomeric configuration as the initial substrate.
The degree of bond cleavage may significantly vary in
the transition states for each step, according to nature
of the enzyme and substrate structure [30.10]. The retaining mechanism for ˛ and ˇ-glycosidases appears to
have similarities and differences, as, for example, the
complementary stereochemical configuration at the glycosyl enzyme intermediate. Speaking in general terms,
evidence for ring distortions in transition states is collected for different biocatalysts. The distortion is driven
by interactions between the enzyme and the substrates
and greatly assists bond cleavage, allowing in-line attack of the nucleophile at the anomeric center. It can
also help the substrate to acquire a conformation closer
to that of the oxocarbenium transition state. Inhibition of these enzymes through the action of compounds
mimicking the structure of the transition states is considered a major area for the development of new therapeutic agents. A collection of detailed studies for the
elucidation of the role of single amino acids in the active
sites, which could be propaedeutic for genetically tailored production of synthetic enzymes (glycosynthases,
see below), can be found in literature [30.13].
Part F | 30.2
can alter or modulate biological functions. Enzymes responsible for the synthesis of glycosidic linkage have
been recognized as transglycosylases and named glycosyltransferases, specifying the glycosyl donor and the
reaction product. These enzymes transfer sugar moieties from the activated donor to specific acceptors,
forming a specific glycosidic bond, and are responsible
in vivo for the synthesis of most cell-surface glycoconjugates, using eight common sugar nucleotides as
activated donors (Leloir pathway). Sugar phosphates
can act as donors for other glycosyltransferases (nonLeloir pathway).
Glycoside hydrolases (glycosidases), represent another widespread group of enzymes involved in the
carbohydrate metabolism, being responsible for the hydrolysis of glycosidic linkages; these enzymes can act
as exo or endoglycosidases and are involved in a series
of biological events of important nature, such as energy
uptake in processes inherent in cell wall metabolism,
in glycan processing during in vivo glycoprotein synthesis, etc. Glycosidases and endoglycosidases are enzymes that are currently leading to new expectations in
research and development due not only to their natural
capability to act on the internal part of an oligosaccharide chain, but also to the possibility of transferring
a whole carbohydrate block to a new acceptor, thus enabling easy reconstruction of complex glycoconjugates
(e.g., glycopeptides, bioactive oligosaccharides, etc.).
In addition, the profitable use of the hydrolytic capabilities of glycosyl hydrolases for mass preparation of
oligosaccharides from natural marine or terrestrial polymers should be mentioned here [30.10].
The stereochemistry of the mechanisms of glycoside hydrolases, analyzed more than 50 years
ago [30.11], allowed the classification of glycoside hydrolases in inverting and retaining enzymes in accord
to the anomeric configuration found in the product with
respect to that in the starting substrate. Very recently,
it has become clear that other mechanisms are evolved,
such as the one based on elimination [30.12].
In the inverting glycoside hydrolases, the glycosidic
substrate, a water molecule, and two carboxylic acid
groups represent the key elements of the chemical machinery within the active site of the enzyme. The two
carboxyl groups acting as general acid and general base
are distant; between them there is a (10:5 Å) value double that of retaining enzymes. This creates room for
the substrate and a water molecule to access the active
site. Hydrolysis occurs via a single displacement mechanism that involves an oxocarbenium ion-like transition
state. The product has the opposite configuration at the
anomeric center with respect to the substrate. If an inverting enzyme catalyzes a transglycosylation reaction
transferring the sugar on an acceptor other than water, the product of this reaction would be the inverted
anomer of the starting glycoside. This transglycosylating activity is never found with these enzymes; in
fact, they would have to be anomerically indiscriminate
to possess such activity. However, inverting enzymes
catalyze oligosaccharide synthesis via reversal of the
hydrolysis reaction; in the presence of a free sugar and
alcohol acceptor, back reaction can produce a glycoside
(see below).
A reaction characterized by a double displacement
mechanism is operative in retaining glycoside hydrolases; two sequential steps can be distinguished in
this case. In the first, the substrate binds the active
site, then a nucleophilic group (aspartic acid, glutamic
acid, or histidine) attacks the anomeric carbon atom.
Protonation of aglycone oxygen by an acid proton,
with concomitant glycosidic bond cleavage and ester
bond formation, leads to the formation of the covalent ester glucosyl-enzyme intermediate, characterized
by anomeric inversion at C1. In the second step, a hydroxyl group from a water molecule (in the case of
the hydrolysis reaction) or from a different residue (for
transglycosylation reactions) attacks the ester bond of
the intermediate with re-inversion of configuration of
the anomeric carbon. As a result, the final product has
the same anomeric configuration as the initial substrate.
The degree of bond cleavage may significantly vary in
the transition states for each step, according to nature
of the enzyme and substrate structure [30.10]. The retaining mechanism for ˛ and ˇ-glycosidases appears to
have similarities and differences, as, for example, the
complementary stereochemical configuration at the glycosyl enzyme intermediate. Speaking in general terms,
evidence for ring distortions in transition states is collected for different biocatalysts. The distortion is driven
by interactions between the enzyme and the substrates
and greatly assists bond cleavage, allowing in-line attack of the nucleophile at the anomeric center. It can
also help the substrate to acquire a conformation closer
to that of the oxocarbenium transition state. Inhibition of these enzymes through the action of compounds
mimicking the structure of the transition states is considered a major area for the development of new therapeutic agents. A collection of detailed studies for the
elucidation of the role of single amino acids in the active
sites, which could be propaedeutic for genetically tailored production of synthetic enzymes (glycosynthases,
see below), can be found in literature [30.13].