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Ionic interaction of positive amino acid residues of fungal hydrophobin RolA with acidic amino acid residues of cutinase CutL1
Author(s) -
Takahashi Toru,
Tanaka Takumi,
Tsushima Yusei,
Muragaki Kimihide,
Uehara Kenji,
Takeuchi Shunsuke,
Maeda Hiroshi,
Yamagata Youhei,
Nakayama Mayumi,
Yoshimi Akira,
Abe Keietsu
Publication year - 2015
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12915
Subject(s) - hydrophobin , quartz crystal microbalance , biochemistry , amino acid , biology , lipase , chemistry , enzyme , adsorption , organic chemistry , gene
Summary Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus A spergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate co‐adipate ( PBSA ), it produces RolA , a hydrophobin, and CutL1 , a PBSA ‐degrading cutinase. Secreted RolA attaches to the surface of the PBSA particles and recruits CutL1 , which then condenses on the particles and stimulates the hydrolysis of PBSA . Here, we identified amino acid residues that are required for the RolA – CutL1 interaction by using site‐directed mutagenesis. We quantitatively analyzed kinetic profiles of the interactions between RolA variants and CutL1 variants by using a quartz crystal microbalance ( QCM ). The QCM analyses revealed that A sp142, A sp171 and G lu31, located on the hydrophilic molecular surface of CutL1 , and H is32 and L ys34, located in the N ‐terminus of RolA , play crucial roles in the RolA – CutL1 interaction via ionic interactions. RolA immobilized on a QCM electrode strongly interacted with CutL1 ( K D = 6.5 nM); however, RolA with CutL1 variants, or RolA variants with CutL1 , showed markedly larger K D values, particularly in the interaction between the double variant RolA ‐ H 32 S / K 34 S and the triple variant CutL1 ‐ E 31 S / D 142 S / D 171 S ( K D = 78.0 nM). We discuss a molecular prototype model of hydrophobin‐based enzyme recruitment at the solid–water interface.