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.
Original language | English |
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Journal | Biocatalysis and Biotransformation |
Volume | 30 |
Issue number | 3 |
Pages (from-to) | 338-352 |
ISSN | 1024-2422 |
DOIs | |
Publication status | Published - 2012 |
Keywords
- alcoholysis reaction
- enzyme activity
- enzyme kinetics
- transglycosylation reaction
- Fungi Plantae (Fungi, Microorganisms, Nonvascular Plants, Plants) - Fungi Imperfecti or Deuteromycetes [15500] Trichoderma reesei species
- arginine 171
- beta-mannanase 70356-42-2 EC 3.2.1.78 N-terminal catalytic module
- butanol 35296-72-1
- galactomannan 11078-30-1
- glutamate 11070-68-1
- glycoside hydrolase 9032-92-2 EC 3.2.2.20
- mannobiose 14417-51-7
- mannose 31103-86-3
- mannotetraose 51327-76-5
- mannotriose 28173-52-6
- methanol 67-56-1
- oligosaccharides
- 10060, Biochemistry studies - General
- 10064, Biochemistry studies - Proteins, peptides and amino acids
- 10068, Biochemistry studies - Carbohydrates
- 10802, Enzymes - General and comparative studies: coenzymes
- 51518, Plant physiology - Enzymes
- Biochemistry and Molecular Biophysics
- mass spectrometry laboratory techniques, spectrum analysis techniques
- site-directed mutagenesis laboratory techniques, genetic techniques
- Enzymology