title: Ionic interaction of positive amino acid residues of fungal hydrophobin RolA with acidic amino acid residues of cutinase CutL1.
abstract: Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus Aspergillus 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 Asp142, Asp171 and Glu31, located on the hydrophilic molecular surface of CutL1, and His32 and Lys34, 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 KD values, particularly in the interaction between the double variant RolA-H32S/K34S and the triple variant CutL1-E31S/D142S/D171S (K(D)  = 78.0 nM). We discuss a molecular prototype model of hydrophobin-based enzyme recruitment at the solid-water interface.
authors.affiliation: Microbial Genomics Laboratory, New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, 981-8555, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Takahashi
authors.forename: Takumi
authors.initials: T
authors.lastname: Tanaka
authors.forename: Yusei
authors.initials: Y
authors.lastname: Tsushima
authors.forename: Kimihide
authors.initials: K
authors.lastname: Muragaki
authors.forename: Kenji
authors.initials: K
authors.lastname: Uehara
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Takeuchi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Maeda
authors.forename: Youhei
authors.initials: Y
authors.lastname: Yamagata
authors.forename: Mayumi
authors.initials: M
authors.lastname: Nakayama
authors.forename: Akira
authors.initials: A
authors.lastname: Yoshimi
authors.forename: Keietsu
authors.initials: K
authors.lastname: Abe
mesh_terms: D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic / Q000378:metabolism*; D001236:Aspergillus oryzae / Q000235:genetics* / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D057927:Hydrophobic and Hydrophilic Interactions; D007477:Ions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011091:Polyesters / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D058525:Quartz Crystal Microbalance Techniques
chemical_list: D024342:Amino Acids, Acidic; D005656:Fungal Proteins; D007477:Ions; D011091:Polyesters; D011108:Polymers; C574545:polybutylene succinate-co-adipate; D002265:Carboxylic Ester Hydrolases; C019952:cutinase