Asp30 and Asp73 of Aspergillus oryzae cutinase CutL1 are involved in the ionic interaction with fungal hydrophobin RolA

Yuki Terauchi yuki.terauchi.s8@dc.tohoku.ac.jp ¹ Yoon-Kyung Kim ¹ Takumi Tanaka ¹ Kei Nanatani ² Akira Yoshimi ³ Toru Takahashi ¹ Keietsu Abe ^1,2,3

¹Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
²Department of Microbial Resources, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan, Sendai, Japan
³Microbial Genomics Laboratory, New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan

When the industrial fungus Aspergillus oryzae is grown with polyesters as a sole carbon source, the fungus co-produces a hydrophobin RolA and an esterase CutL1. RolA attached to polyesters specifically recruits CutL1 and consequently promotes hydrolysis of polyesters by CutL1. The mechanism of its recruitment is attributed to the ionic interaction between positively charged residues (H32, K34) of RolA and negatively charged residues (E31, D142, D171) of CutL1. The K_D for the interaction of RolA with the CutL1-E31S/D142S/D171S was considerably higher than that for its interaction with wild-type CutL1. In the presence of 250 mM NaCl, both K_D values were similar, suggesting that some additional charged residues in CutL1 are involved in the CutL1—RolA interaction besides E31, D142 and D171. In this study, we investigated whether D30 and D73 of CutL1 are also involved in the CutL1—RolA interaction. First, we compared amino acid sequences of CutL1 and CutL1 orthologs, and analyzed CutL1 3D-model, leading to prediction of D30 and D73 as the candidate residues involved in RolA-CutL1 interaction. Next, we purified CutL1-D30S, CutL1-D73S, CutL1-D30S/E31S/D142S/D171S and CutL1-E31S/D73S/D142S/D171S, and measured Circular Dichroism (CD) spectra of them to confirm their structures. To analyze the kinetics of binding of the CutL1 mutants to RolA, we used a Quartz Crystal Microbalance (QCM) and calculated K_D. The QCM approach revealed that the K_D values of the CutL1 single mutants to RolA were higher than that of wild-type CutL1 to RolA, and the K_D values of CutL1 quadruple mutants to RolA were higher than that of CutL1 E31S/D142S/D171S. We conclude that D30 and D73 are important for CutL1–RolA interaction. These results also imply that CutL1 3D-model is useful for prediction of amino acid residues that are involved in the interaction with RolA despite little conservation of the residues among CutL1 orthologs.