Abstract
A novel oligoalginate lyase from a marine bacterium, Sphingomonas sp. strain MJ-3, exhibited a unique alginate degradation activity that completely depolymerizes alginate to monomers through the formation of oligomers. In order to reveal the reason why MJ-3 oligoalginate can exhibit both endolytic and exolytic alginate lyase activities, ten mutants were developed and characterized on the basis of homology modeling. When the recombinant cell lysates containing the mutated proteins of MJ-3 oligoalginate lyase were allowed to react with alginate, the Asn177Ala, His178Ala, Tyr234Phe, His389Ala, and Tyr426Phe mutants showed reduced oligoalginate lyase activity, whereas the Arg236Ala mutant exhibited endolytic activity. Interestingly, the overexpressed Arg236Ala protein (79.6 kDa) was proteolytically cleaved into two fragments, i.e., the N-terminal 32.0-kDa and the C-terminal 47.6-kDa fragments. Both the purified N-terminal and C-terminal fragments showed endolytic lyase activity. They preferentially degraded a heteropolymeric (polyMG) block than poly-β-D-mannuronate (polyM) or poly-α-L-guluronate (polyG) blocks. These results suggest that the oligoalginate lyase activity of MJ-3 enzyme is derived from the cooperative interaction between the N- and C-terminal endolytic alginate lyase domains in the intact enzyme.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Draget KI, Smidsrød O, Skjåk-Bræk G (2005) Alginates from Algae. Biopolymers Online, 6. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Gacesa P (1998) Bacterial alginate biosynthesis—recent progress and future prospects. Microbiology 144:1133–1143
Hashimoto W, Miyake O, Momma K, Kawai S, Murata K (2000) Molecular identification of oligoalginate lyase of Sphingomonas sp. Strain A1 as one of the enzymes required for complete depolymerization of alginate. J Bacteriol 182:4572–4577
Haug A, Larsen B, Smidsrot O (1966) A study of the constitution of alginic acid by partial acid hydrolysis. Acta Chem Scand 20:183–190
Iwamoto M, Kurachi M, Nakashima T, Kim D, Yamaguchi K, Oda T, Iwamoto Y, Muramatsu T (2005) Structure–activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells. FEBS Lett 579:4423–4429
Kam N, Park YJ, Lee EY, Kim HS (2011) Molecular identification of a polyM-specific alginate lyase from Pseudomonas sp. strain KS-408 for degradation of glycosidic linkages between two mannuronates or mannuronate and guluronate in alginate. Can J Microbiol 57:1032–1041
Kim DE, Lee EY, Kim HS (2009) Cloning and characterization of alginate lyase from a marine bacterium Streptomyces sp. ALG-5. Mar Biotechnol 11:10–16
Kim HS, Lee C-G, Lee EY (2011) Alginate lyase: structure, property and application. Biotechnol Bioprocess Eng 16:843–851
Lee SI, Choi SH, Lee EY, Kim HS (2012) Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomonas maltophilia KJ-2. Appl Microbiol Biotechnol 95:1643–1653
Marti-Renom MA, Stuart AC, Fiser A, Sanchez R, Melo F, Sali A (2000) Comparative protein structure modelling of genes and genomes. Annu Rev Biophys Biomol Struct 29:291–325
Mikami B, Ban M, Suzuki S, Yoon HJ, Miyake O, Yamasaki M, Ogura K, Maruyama Y, Hashimoto W, Murata K (2012) Induced-fit motion of a lid loop involved in catalysis in alginate lyase A1-III. Acta Crystallogr D Biol Crystallogr 68:1207–1216
Miyake O, Hashimoto W, Murata K (2003) An exotype alginate lyase in Sphingomonas sp. A1: Overexpression in Escherichia coli, purification, and characterization of alginate lyase IV (A1-IV). Protein Expr Purif 29:33–41
Momma K, Okamoto M, Mishima Y, Mori S, Hashimoto W, Murata K (2000) A novel bacterial ATP-binding cassette transporter system that allows uptake of macromolecules. J Bacteriol 182:3998–4004
Murata K, Inose T, Hisano T, Abe S, Yonemoto Y, Yamashita T, Takagi M, Sakaguchi K, Kimura A, Imanaka T (1993) Bacterial alginate lyase: enzymology, genetics and application. J Ferment Bioeng 76:427–437
Ochiai A, Hashimoto W, Murata K (2006) A biosystem for alginate metabolism in Agrobacterium tumefaciens strain C58: Molecular identification of Atu3025 as an exotype family PL-15 alginate lyase. Res Microbiol 157:642–649
Ochiai A, Yamasaki M, Mikami B, Hashimoto W, Murata K (2010) Crystal structure of exotype alginate lyase Atu3025 from Agrobacterium tumefaciens. J Biol Chem 285:24519–24528
Park HH, Kam N, Lee EY, Kim HS (2012) Cloning and characterization of a novel oligoalginate lyase from a newly isolated bacterium Sphingomonas sp. MJ-3. Mar Biotechnol 14:189–202
Park D, Jagtap S, Nair SK (2014a) Structure of a PL17 family alginate lyase demonstrates functional similarities among exotype depolymerases. J Biol Chem 289:8645–8655
Park YJ, Chu YJ, Shin YH, Lee EY, Kim HS (2014b) Molecular cloning and characterization of a novel acetylalginate esterase gene in alg operon from Sphingomonas sp. MJ-3. Appl Microbiol Biotechnol 98:2145–2154
Ryu M, Lee EY (2011) Saccharification of alginate by using exolytic oligoalginate lyase from marine bacterium Sphingomonas sp. MJ-3. J Ind Eng Chem 17:853–858
Shaya D, Tocilj A, Li Y, Myette J, Venkataraman G, Sasisekharan R, Cygler M (2006) Crystal structure of heparinase II from Pedobacter heparinus and its complex with a disaccharide product. J Biol Chem 281:15525–15535
Shin JW, Lee OK, Park HH, Kim HS, Lee EY (2015) Molecular characterization of a novel oligoalginate lyase consisting of AlgL- and heparinase II/III-like domains from Stenotrophomonas maltophilia KJ-2 and its application to alginate saccharification. Korean J Chem Eng 32:917–924
Takeda H, Yoneyama F, Kawai S, Hashimoto W, Murata K (2011) Bioethanol production from marine biomass alginate by metabolically engineered bacteria. Energy Environ Sci 4:2575–2581
Weissbach A, Hurwitz J (1959) The formation of 2-keto-3-deoxyheptonic acid in extracts of Escherichia. J Biol Chem 234:705–709
Wong TY, Preston LA, Schiller NL (2000) Alginate lyase: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Annu Rev Microbiol 54:289–340
Yonemoto Y, Tanaka H, Hisano T, Sakaguchi K, Abe S, Yamashita T, Kimura A, Murata K (1993) Bacterial alginate lyase gene: nucleotide sequence and molecular route for generation of alginate lyase species. J Ferment Bioeng 75:336–342
Yoon H-J, Hashimoto W, Miyake O, Murata K, Mikami B (2001) Crystal structure of alginate lyase A1-III complexed with trisaccharide product at 2.0 Å resolution. J Mol Biol 307:9–16
Acknowledgments
This work was supported by the Ministry of Oceans and Fisheries, Republic of Korea. This work was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2012R1A1A2008647).
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Kim, H.S., Chu, Y.J., Park, CH. et al. Site-Directed Mutagenesis-Based Functional Analysis and Characterization of Endolytic Lyase Activity of N- and C-Terminal Domains of a Novel Oligoalginate Lyase from Sphingomonas sp. MJ-3 Possessing Exolytic Lyase Activity in the Intact Enzyme. Mar Biotechnol 17, 782–792 (2015). https://doi.org/10.1007/s10126-015-9658-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10126-015-9658-4