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

Abstract

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 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 (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.

Keywords::

• β-mannanase
• enzyme kinetics
• transglycosylation
• site-directed mutagenesis
• isotope labelling
• alcoholysis

Acknowledgments

Dr. Eila Cedergren (Biochemistry and Structural biology, Lund University) is thanked especially for the help and discussions involving the structural analysis of the R171K substitution. Magnus Nilsson is thanked for technical assistance in part of the mutagenesis.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.

The Swedish Research Council (VR), and the Crafoord foundation are thanked for research grants to Henrik Stålbrand.