Structural and biochemical characterisation of a novel alginate lyase from Paenibacillus sp. str. FPU-7

• Published in: Scientific reports 2019-10, Vol.9 (1), p.14870-14, Article 14870
• Main Authors: Itoh, Takafumi, Nakagawa, Emi, Yoda, Moe, Nakaichi, Akari, Hibi, Takao, Kimoto, Hisashi
• Format: Article
• Language: English
• Subjects: 631/326/41/2536; 631/45/535/1266; 631/45/607; 631/45/72/1205; Acetic acid; Alginate lyase; Alginic acid; Alginic Acid - chemistry; Alginic Acid - metabolism; Amino acid sequence; Amino acids; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Carbon dioxide; Catalysis; Catalytic Domain; Cations; Cations, Divalent; Cloning, Molecular; Cobalt; Cobalt - chemistry; Cobalt - metabolism; Crystal structure; Crystallography, X-Ray; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Humanities and Social Sciences; Kinetics; Magnesium; Magnesium - chemistry; Magnesium - metabolism; Manganese - chemistry; Manganese - metabolism; Molecular Docking Simulation; Molecular Weight; multidisciplinary; Paenibacillus; Paenibacillus - chemistry; Paenibacillus - enzymology; Paenibacillus - genetics; pH effects; Polysaccharide-Lyases - chemistry; Polysaccharide-Lyases - genetics; Polysaccharide-Lyases - metabolism; Polysaccharides; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Science; Science (multidisciplinary); Substrate Specificity; Thermodynamics
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-51006-1

A novel alginate lyase, PsAly, with a molecular mass of 33 kDa and whose amino acid sequence shares no significant similarity to other known proteins, was biochemically and structurally characterised from Paenibacillus sp. str. FPU-7. The maximum PsAly activity was obtained at 65 °C, with an optimum pH of pH 7–7.5. The activity was enhanced by divalent cations, such as Mg 2+ , Mn 2+ , or Co 2+ , and inhibited by a metal chelator, ethylenediaminetetraacetic acid. The reaction products indicated that PsAly is an endolytic enzyme with a preference for polymannuronate. Herein, we report a detailed crystal structure of PsAly at a resolution of 0.89 Å, which possesses a β-helix fold that creates a long cleft. The catalytic site was different from that of other polysaccharide lyases. Site-directed mutational analysis of conserved residues predicted Tyr184 and Lys221 as catalytic residues, abstracting from the C5 proton and providing a proton to the glycoside bond, respectively. One cation was found to bind to the bottom of the cleft and neutralise the carboxy group of the substrate, decreasing the p K a of the C5 proton to promote catalysis. Our study provides an insight into the structural basis for the catalysis of alginate lyases and β-helix polysaccharide lyases.