respective non-alkaline counterparts, the alkaline active hemicellulases display a
high percentage composition of acidic amino acids, fewer solvent exposed polar
residues, acidic surfaces, and fewer solvent exposed alkali labile residues [135, 140,
144].
Three different types of active site topological arrangements are recognized in
GHs [146]. A tunnel is uniquely suited for processive exo-attack as in the case of
β-glucosidase. Another active site topology is a cleft which allows endo-attack like
endo-xylanases. The third topology is a crater/pocket that mostly fits for degradation
of substrates by end-on-attack like GH8 xylanases. GH10 xylanases have clefts
in the large radius face of their structure. At least in this group of enzymes, the
cleft topology does not seem conserved. For instance, GH10 xylanases from
P. simplicissimum (PDB 1B30) and T. aurantiacus (PDB 1K6A) have very
shallow clefts, while xylanases of B. halodurans S7 (PDB 2UWF) and
G. stearothermophilus (PDB 1HIZ) have relatively deep clefts [135]. The variation
in the cleft topology can affect enzyme properties. As aforementioned, generally,
GH10 xylanases show broader substrate specificity when compared to GH11
xylanases. One of the possible reasons for the difference in the substrate specificity
could be the depth of their active site clefts [147]. GH11 xylanases have deeper
cleft than GH10 xylanases. However, there are some xylan-specific GH10 xylanases
such as the alkaline active xylanase from B. halodurans S7. Such GH10 xylanases
have deeper catalytic cleft which might be the reason behind their substrate
specificity [135].
Analysis of the GH5 mannanases three-dimensional structures has indicated the
tendency of increasing structural compactness with an increase in the enzymes’
optimum pH for activity [140]. There seems to be rearrangement of secondary
structures around the active site region, which distinctly shape the catalytic cleft of
the alkaline active mannanases. This unique microenvironment created by the
rearrangement of loops, helices, and β-strands affects the catalytic amino acids
protonation state, which determines the optimum pH for the mannanase activity.
Mutating residues that exist in the microenvironment can affect the pH profile of
mannanases, and indeed this has been proven [140].
5.5 Mechanism of Catalysis
There is a wide variety of glycosidic bonds in organic molecules. These bonds are
vital in formation of carbohydrate polymers such as xylan and mannan. There exists
a range of enzymes whose function is to cleave these bonds. Generally, hydrolysis of
this bond by an enzyme carried out with one of the two stereochemical outcomes, net
inversion or retention of the anomeric configuration. Based on this, glycosidases
(including xylanases and mannanases) are classified as inverting or retaining. The
inverting mechanism involves transition states and a pair of carboxylic acids at the
active site, and the reaction occurs via a single displacement mechanism wherein one
carboxylic acid acts as a general base and the other as a general acid. The average
268
G. Mamo
high percentage composition of acidic amino acids, fewer solvent exposed polar
residues, acidic surfaces, and fewer solvent exposed alkali labile residues [135, 140,
144].
Three different types of active site topological arrangements are recognized in
GHs [146]. A tunnel is uniquely suited for processive exo-attack as in the case of
β-glucosidase. Another active site topology is a cleft which allows endo-attack like
endo-xylanases. The third topology is a crater/pocket that mostly fits for degradation
of substrates by end-on-attack like GH8 xylanases. GH10 xylanases have clefts
in the large radius face of their structure. At least in this group of enzymes, the
cleft topology does not seem conserved. For instance, GH10 xylanases from
P. simplicissimum (PDB 1B30) and T. aurantiacus (PDB 1K6A) have very
shallow clefts, while xylanases of B. halodurans S7 (PDB 2UWF) and
G. stearothermophilus (PDB 1HIZ) have relatively deep clefts [135]. The variation
in the cleft topology can affect enzyme properties. As aforementioned, generally,
GH10 xylanases show broader substrate specificity when compared to GH11
xylanases. One of the possible reasons for the difference in the substrate specificity
could be the depth of their active site clefts [147]. GH11 xylanases have deeper
cleft than GH10 xylanases. However, there are some xylan-specific GH10 xylanases
such as the alkaline active xylanase from B. halodurans S7. Such GH10 xylanases
have deeper catalytic cleft which might be the reason behind their substrate
specificity [135].
Analysis of the GH5 mannanases three-dimensional structures has indicated the
tendency of increasing structural compactness with an increase in the enzymes’
optimum pH for activity [140]. There seems to be rearrangement of secondary
structures around the active site region, which distinctly shape the catalytic cleft of
the alkaline active mannanases. This unique microenvironment created by the
rearrangement of loops, helices, and β-strands affects the catalytic amino acids
protonation state, which determines the optimum pH for the mannanase activity.
Mutating residues that exist in the microenvironment can affect the pH profile of
mannanases, and indeed this has been proven [140].
5.5 Mechanism of Catalysis
There is a wide variety of glycosidic bonds in organic molecules. These bonds are
vital in formation of carbohydrate polymers such as xylan and mannan. There exists
a range of enzymes whose function is to cleave these bonds. Generally, hydrolysis of
this bond by an enzyme carried out with one of the two stereochemical outcomes, net
inversion or retention of the anomeric configuration. Based on this, glycosidases
(including xylanases and mannanases) are classified as inverting or retaining. The
inverting mechanism involves transition states and a pair of carboxylic acids at the
active site, and the reaction occurs via a single displacement mechanism wherein one
carboxylic acid acts as a general base and the other as a general acid. The average
268
G. Mamo