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The role of subsite +2 of the Trichoderma reesei beta-mannanase TrMan5A in hydrolysis and transglycosylation

Rosengren, Anna LU ; Hagglund, Per; Anderson, Lars; Pavon-Orozco, Patricia; Peterson Wulff, Ragna LU ; Nerinckx, Wim and Stålbrand, Henrik LU (2012) In Biocatalysis and Biotransformation 30(3). p.338-352
Abstract
The N-terminal catalytic module of beta-mannanase TrMan5A from the filamentous fungus Trichoderma reesei is classified into family 5 of glycoside hydrolases. It is further classified in clan A with a (beta/alpha)(8) barrel configuration and has two catalytic glutamates (E169 and E276). It has at least five other residues conserved in family 5. Sequence alignment revealed that an arginine (R171 in TrMan5A) is semi-conserved among beta-mannanases in family 5. In a previously published mannobiose complex structure, this residue is positioned in hydrogen bonding distance from the C2 hydroxyl group of the mannose residue bound at the +2 subsite. To study the function of R171, mutants of this residue were constructed. The results show that... (More)
The N-terminal catalytic module of beta-mannanase TrMan5A from the filamentous fungus Trichoderma reesei is classified into family 5 of glycoside hydrolases. It is further classified in clan A with a (beta/alpha)(8) barrel configuration and has two catalytic glutamates (E169 and E276). It has at least five other residues conserved in family 5. Sequence alignment revealed that an arginine (R171 in TrMan5A) is semi-conserved among beta-mannanases in family 5. In a previously published mannobiose complex structure, this residue is positioned in hydrogen bonding distance from the C2 hydroxyl group of the mannose residue bound at the +2 subsite. To study the function of R171, mutants of this residue were constructed. The results show that arginine 171 is important for substrate binding and transglycosylation. A mutant of TrMan5A with the substitution R171K displayed retained activity on polymeric galactomannan but reduced activity on oligosaccharides due to an increase of K-m. While the wild-type enzyme produces mannobiose as dominant product from mannotetraose the R171K mutant shows an altered product profile, producing mannotriose and mannose. The cleavage pattern of mannotetraose was analysed with a method using isotope labelled water ((H2O)-O-18) and mass spectrometry which showed that the preferred productive binding mode of mannotetraose was shifted from subsite -2 to +2 in the wild-type to subsite -3 to +1 in the R171K mutant. Significant differences in product formation after manno-oligosaccharide incubation showed that the wild-type enzyme can perform transglycosylation on to saccharide acceptors while the R171K mutant cannot, likely due to loss of acceptor affinity. Interestingly, both enzymes show the ability to perform alcoholysis reactions with methanol and butanol, forming new beta-linked glyco-conjugates. Furthermore, it appears that the wild-type enzyme produces mainly mannobiose conjugates using M-4 as substrate, while in contrast the R171K mutant produces mainly mannotriose conjugates, due to the altered subsite binding. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
beta-mannanase, enzyme kinetics, transglycosylation, site-directed, mutagenesis, isotope labelling, alcoholysis
in
Biocatalysis and Biotransformation
volume
30
issue
3
pages
338 - 352
publisher
Taylor & Francis
external identifiers
  • wos:000304750500008
  • scopus:84861760915
ISSN
1024-2422
DOI
10.3109/10242422.2012.674726
language
English
LU publication?
yes
id
a664f945-18b9-4feb-b626-f93d788fa102 (old id 2903222)
date added to LUP
2012-07-24 10:10:29
date last changed
2017-08-20 03:12:08
@article{a664f945-18b9-4feb-b626-f93d788fa102,
  abstract     = {The N-terminal catalytic module of beta-mannanase TrMan5A from the filamentous fungus Trichoderma reesei is classified into family 5 of glycoside hydrolases. It is further classified in clan A with a (beta/alpha)(8) barrel configuration and has two catalytic glutamates (E169 and E276). It has at least five other residues conserved in family 5. Sequence alignment revealed that an arginine (R171 in TrMan5A) is semi-conserved among beta-mannanases in family 5. In a previously published mannobiose complex structure, this residue is positioned in hydrogen bonding distance from the C2 hydroxyl group of the mannose residue bound at the +2 subsite. To study the function of R171, mutants of this residue were constructed. The results show that arginine 171 is important for substrate binding and transglycosylation. A mutant of TrMan5A with the substitution R171K displayed retained activity on polymeric galactomannan but reduced activity on oligosaccharides due to an increase of K-m. While the wild-type enzyme produces mannobiose as dominant product from mannotetraose the R171K mutant shows an altered product profile, producing mannotriose and mannose. The cleavage pattern of mannotetraose was analysed with a method using isotope labelled water ((H2O)-O-18) and mass spectrometry which showed that the preferred productive binding mode of mannotetraose was shifted from subsite -2 to +2 in the wild-type to subsite -3 to +1 in the R171K mutant. Significant differences in product formation after manno-oligosaccharide incubation showed that the wild-type enzyme can perform transglycosylation on to saccharide acceptors while the R171K mutant cannot, likely due to loss of acceptor affinity. Interestingly, both enzymes show the ability to perform alcoholysis reactions with methanol and butanol, forming new beta-linked glyco-conjugates. Furthermore, it appears that the wild-type enzyme produces mainly mannobiose conjugates using M-4 as substrate, while in contrast the R171K mutant produces mainly mannotriose conjugates, due to the altered subsite binding.},
  author       = {Rosengren, Anna and Hagglund, Per and Anderson, Lars and Pavon-Orozco, Patricia and Peterson Wulff, Ragna and Nerinckx, Wim and Stålbrand, Henrik},
  issn         = {1024-2422},
  keyword      = {beta-mannanase,enzyme kinetics,transglycosylation,site-directed,mutagenesis,isotope labelling,alcoholysis},
  language     = {eng},
  number       = {3},
  pages        = {338--352},
  publisher    = {Taylor & Francis},
  series       = {Biocatalysis and Biotransformation},
  title        = {The role of subsite +2 of the Trichoderma reesei beta-mannanase TrMan5A in hydrolysis and transglycosylation},
  url          = {http://dx.doi.org/10.3109/10242422.2012.674726},
  volume       = {30},
  year         = {2012},
}