title: Biochemical and structural characterization of a keratin-degrading M32 carboxypeptidase from Fervidobacterium islandicum AW-1.
abstract: Comparative genomics of the keratin-degrading extremophilic eubacterium Fervidobacterium islandicum AW-1 and the closely related Fervidobacterium nodosum with no keratinolytic activity suggested that the FIAW1_1600 gene encoding a carboxypeptidase (CP) plays an important role in keratin degradation. The presumptive 489 amino acid sequence of the gene showed a conserved HEXXH motif with low levels of sequence identity (<38%) to reported thermostable M32 CPs. To identify its functional role, the FIAW1_1600 gene was overexpressed in Escherichia coli, and the recombinant enzyme was purified and characterized in detail. F. islandicum AW-1 CP (FisCP) formed a homodimer with a molecular mass of 107 kDa, and its apoenzyme exhibited maximal activity at 80 °C and pH 7.0 in the presence of Co(2+). This metalloenzyme mainly cleaved the C-termini of peptides with a basic amino acid sequence. The crystal structure of FisCP at 2.2 Å resolution showed high levels of structural similarities (root-mean-square deviations of <1.7 Å) to those of other M32 CP homologs. Remarkably, the enzyme significantly enhanced the degradation of native chicken feathers. This study suggests that FisCP, a keratinolytic member of the thermostable M32 CP family, plays an important role in keratin degradation for cellular metabolism in F. islandicum AW-1.
authors.affiliation: School of Applied Biosciences, Kyungpook National University, Daegu 702-701, South Korea.
authors.forename: Yong-Jik
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: Department of Cellular and Molecular Medicine, Chosun University School of Medicine, Gwangju 501-759, South Korea.
authors.forename: Immanuel
authors.initials: I
authors.lastname: Dhanasingh
authors.affiliation: School of Applied Biosciences, Kyungpook National University, Daegu 702-701, South Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Ahn
authors.affiliation: School of Applied Biosciences, Kyungpook National University, Daegu 702-701, South Korea.
authors.forename: Hyeon-Su
authors.initials: HS
authors.lastname: Jin
authors.affiliation: Department of Cellular and Molecular Medicine, Chosun University School of Medicine, Gwangju 501-759, South Korea.
authors.forename: Jin Myung
authors.initials: JM
authors.lastname: Choi
authors.affiliation: Department of Cellular and Molecular Medicine, Chosun University School of Medicine, Gwangju 501-759, South Korea. Electronic address: sunglee@chosun.ac.kr.
authors.forename: Sung Haeng
authors.initials: SH
authors.lastname: Lee
authors.affiliation: School of Applied Biosciences, Kyungpook National University, Daegu 702-701, South Korea. Electronic address: leehicam@knu.ac.kr.
authors.forename: Dong-Woo
authors.initials: DW
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry* / Q000648:ultrastructure*; D002384:Catalysis; D004789:Enzyme Activation; D004795:Enzyme Stability; D007633:Keratins / Q000737:chemistry* / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D007633:Keratins; D002268:Carboxypeptidases
keywords: Extremophiles; Fervidobacterium islandicum AW-1; Keratin degradation; M32 carboxypeptidase; Structure