Engineering Fungal Expression Systems: Recombinant Expression of Two Class I a-1, 2-Mannosidases from the Filamentous Fungus Aspergillus nidulans - Biotechnology of Fungal Genes - page 280

those of P. citrinum , S. cerevisiae and T. reesei enzymes would predict the
ability of the A. nidulans enzymes to cleave Man 9 GlcNAc 2 to Man 5 GlcNAc 2 .
Critical amino acids in the P. citrinum α-1,2-mannosidase are different than
those found at the same position in the S. cerevisiae enzyme. These changes
were generally directed towards smaller side chains which increased the
size and accessibility of the binding pocket and these differences could
partially explain the difference in substrate specifi city of these enzymes.
For instance, one of the most critical amino acid changes between the S.
cerevisiae and P. citrinum enzymes is the replacement of an arginine residue,
Arg 273 in the yeast polypeptide, with a glycine residue, Gly 265 in the fungal
polypeptide (Lobsanov et al. 2002). In the yeast enzyme, this amino acid
has been shown to interact with several mannose residues in the binding
pocket. Mutation of Arg 273 in yeast to a Leu residue altered the specifi city
of the enzyme such that it could cleave Man 9 GlcNA 2 to Man 5 GlcNAc 2
(Romero et al. 2000). That this amino acid is changed in P. citrinum to a
glycine, which has no side chain, suggests that this amino acid is critical
in allowing greater accessibility of the substrate to the cleavage domain
of the enzyme, thus changing the specifi city of the enzyme. This specifi c
amino acid change is also observed in the A. nidulans α-1,2-mannosidase
IB and α-1,2-mannosidase IC enzymes—the corresponding amino acids
are glycine, Gly 257 and Gly 333 respectively hence both of these enzymes
would be expected to process N-glycans to the Man 5 GlcNac 2 form. Other
differences between the S. cerevisiae and P. citrinum polypeptides are also
seen in the α-1,2-mannosidase IB and α-1,2-mannosidase IC polypeptides,
such as the replacement of Arg 269 in the yeast polypeptide to Ser in the
fungal polypeptides. These changes help to explain the ability of the A.
nidulans α-1,2-mannosidase IB and α-1,2-mannosidase IC enzymes to cleave
Man 9 GlcNAc 2 fully to Man 5 GlcNAc 2 .
When Man 9 GlcNAc 2 digestions were analyzed over time, we were
able to further characterize the rate at which intermediates were produced
during the course of these reactions ( Fig. 2) . Both the α-1,2-mannosidase IB
and IC enzyme rapidly degraded Man 9 GlcNAc 2 to Man 7 GlcNAc 2 , but then
proceeded much more slowly to Man 6 GlcNAc 2 and on to Man 5 GlcNAc 2 . This
led to the accumulation of Man 7 GlcNAc 2 and Man 6 GlcNAc 2 intermediates
during the digestion process. These were eventually converted to
Man 5 GlcNAc 2 . Interestingly, the α-1,2-mannosidase IC enzyme appeared
to convert these intermediates to Man 5 GlcNAc 2 more rapidly than the
α-1,2-mannosidase IB enzyme, even though the α-1,2-mannosidase IB
had a higher specifi c activity towards the synthetic substrate Man-α-1,2-
Man-OCH 3 . This difference may be related to certain structural differences
which limit the access of these intermediates within the binding pocket to
the catalytic region of the enzyme.