The role of subsite 2 of the Trichoderma reesei β-mannanase TrMan5A in hydrolysis and transglycosylation
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The role of subsite 2 of the Trichoderma reesei β-mannanase TrMan5A in hydrolysis and transglycosylation. / Rosengren, Anna; Hägglund, Per; Anderson, Lars; Pavon-Orozco, Patricia; Peterson-Wulff, Ragna; Nerinckx, Wim; Stålbrand, Henrik.
I: Biocatalysis and Biotransformation, Bind 30, Nr. 3, 01.05.2012, s. 338-352.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - The role of subsite 2 of the Trichoderma reesei β-mannanase TrMan5A in hydrolysis and transglycosylation
AU - Rosengren, Anna
AU - Hägglund, Per
AU - Anderson, Lars
AU - Pavon-Orozco, Patricia
AU - Peterson-Wulff, Ragna
AU - Nerinckx, Wim
AU - Stålbrand, Henrik
PY - 2012/5/1
Y1 - 2012/5/1
N2 - The N-terminal catalytic module of β-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 (β/α)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 β-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 Km. 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 (H218O) 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 β-linked glyco-conjugates. Furthermore, it appears that the wild-type enzyme produces mainly mannobiose conjugates using M4 as substrate, while in contrast the R171K mutant produces mainly mannotriose conjugates, due to the altered subsite binding.
AB - The N-terminal catalytic module of β-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 (β/α)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 β-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 Km. 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 (H218O) 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 β-linked glyco-conjugates. Furthermore, it appears that the wild-type enzyme produces mainly mannobiose conjugates using M4 as substrate, while in contrast the R171K mutant produces mainly mannotriose conjugates, due to the altered subsite binding.
KW - β-mannanase
KW - Alcoholysis
KW - Enzyme kinetics
KW - Isotope labelling
KW - Site-directed mutagenesis
KW - Transglycosylation
UR - http://www.scopus.com/inward/record.url?scp=84861760915&partnerID=8YFLogxK
U2 - 10.3109/10242422.2012.674726
DO - 10.3109/10242422.2012.674726
M3 - Journal article
AN - SCOPUS:84861760915
VL - 30
SP - 338
EP - 352
JO - Biocatalysis and Biotransformation
JF - Biocatalysis and Biotransformation
SN - 1024-2422
IS - 3
ER -
ID: 240159990