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• W2974863485 abstract "Alginate is a linear polysaccharide from brown algae consisting of 1,4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome–associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel β-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel β-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2–7 from alginate and polyM, which were further degraded to di- and trisaccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC–electrospray ionization–MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes. Alginate is a linear polysaccharide from brown algae consisting of 1,4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome–associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel β-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel β-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2–7 from alginate and polyM, which were further degraded to di- and trisaccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC–electrospray ionization–MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes. Alginates are linear anionic polysaccharides present in the cell walls of brown seaweeds. They are composed of blocks of 1,4-linked β-d-mannuronic acid (M), 2The abbreviations used are: Mβ-d-mannuronic acidBcelPL6family 6 polysaccharide lyase from B. cellulosilyticus CRE21Δ4-deoxy-l-erythro-hex-4-enopyranosyluronic acidDP3GDP4G, DP3M, DP4M, tri- or tetrasaccharides of α-l-guluronic acid or β-d-mannuronic acidDP8Mocta-mannuronic acidLC-ESI-MSliquid chromatography–electron spray ionization–mass spectrometryHGMhuman gut microbiotaGα-l-guluronic acidM_nnumber average molecular weightM_wweight average molecular weightPDBProtein Data BankPLpolysaccharide lyasePL6polysaccharide lyase family 6polyGpoly-α-l-guluronic acidpolyMpoly-β-d-mannuronic acidpolyMGalternating or random β-d-mannuronic acid and α-l-guluronic acid polymerSECsize-exclusion chromatographyDSCdifferential scanning calorimetryACNacetonitrilePULpolysaccharide utilization loci. its C-5 epimer α-l-guluronic acid (G), and of both M and G arranged in alternating or random order (Fig. 1A) (1Haug A. Larsen B. Smidsrod O. Studies on sequence of uronic acid residues in alginic acid.Acta Chem. Scand. 1967; 21: 691-70410.3891/acta.chem.scand.21-0691Crossref Google Scholar, 2Aarstad O.A. Tøndervik A. Sletta H. Skjåk-Bræk G. Alginate sequencing: an analysis of block distribution in alginates using specific alginate degrading enzymes.Biomacromolecules. 2012; 13 (22148348): 106-11610.1021/bm2013026Crossref PubMed Scopus (86) Google Scholar). Alginates are hydrocolloids and serve as gelling and stabilizing agents in food and pharmaceutical products (sodium alginate ref. no. 00148) (4Yao M. Wu J. Li B. Xiao H. McClements D.J. Li L. Microencapsulation of Lactobacillus salivarious Li01 for enhanced storage viability and targeted delivery to gut microbiota.Food Hydrocoll. 2017; 72: 228-23610.1016/j.foodhyd.2017.05.033Crossref Scopus (72) Google Scholar). Moreover, alginates and alginate oligosaccharides have applications in the biomedicine and health sectors (5Xu X. Bi D. Wan M. Characterization and immunological evaluation of low-molecular-weight alginate derivatives.Curr. Top. Med. Chem. 2016; 16 (26311423): 874-88710.2174/1568026615666150827101239Crossref PubMed Scopus (24) Google Scholar, 6Odunsi S.T. Vázquez-Roque M.I. Camilleri M. Papathanasopoulos A. Clark M.M. Wodrich L. Lempke M. McKinzie S. Ryks M. Burton D. Zinsmeister A.R. Effect of alginate on satiation, appetite, gastric function, and selected gut satiety hormones in overweight and obesity.Obesity. 2010; 18 (19960001): 1579-158410.1038/oby.2009.421Crossref PubMed Scopus (62) Google Scholar, 7Okolie C.L. Subin S.R. Udenigwe C.C. Aryee A.N.A. Mason B. Prospects of brown seaweed polysaccharides (BSP) as prebiotics and potential immunomodulators.J. Food Biochem. 2017; 41: 1-1210.1111/jfbc.12392Crossref Scopus (58) Google Scholar). Biofilms produced by some terrestrial bacteria, e.g. Azotobacter vinelandii and Pseudomonas aeruginosa, contain alginates O-acetylated on C2 and C3 in the M blocks with low G content (8Urtuvia V. Maturana N. Acevedo F. Peña C. Díaz-Barrera A. Bacterial alginate production: an overview of its biosynthesis and potential industrial production.World J. Microbiol. Biotechnol. 2017; 33 (28988302): 19810.1007/s11274-017-2363-xCrossref PubMed Scopus (70) Google Scholar). β-d-mannuronic acid family 6 polysaccharide lyase from B. cellulosilyticus CRE21 4-deoxy-l-erythro-hex-4-enopyranosyluronic acid DP4G, DP3M, DP4M, tri- or tetrasaccharides of α-l-guluronic acid or β-d-mannuronic acid octa-mannuronic acid liquid chromatography–electron spray ionization–mass spectrometry human gut microbiota α-l-guluronic acid number average molecular weight weight average molecular weight Protein Data Bank polysaccharide lyase polysaccharide lyase family 6 poly-α-l-guluronic acid poly-β-d-mannuronic acid alternating or random β-d-mannuronic acid and α-l-guluronic acid polymer size-exclusion chromatography differential scanning calorimetry acetonitrile polysaccharide utilization loci. Humans lack alginate-degrading enzymes, but certain gut bacteria, e.g. strains of the commensal Bacteroides ovatus, Bacteroides xylanisolvens, and Bacteroides thetaiotaomicron, can grow on and ferment alginate in vitro to form healthy and beneficial short-chain fatty acids (9Akiyama H. Endo T. Nakakita R. Murata K. Yonemoto Y. Okayama K. Effect of depolymerized alginates on the growth of bifidobacteria.Biosci. Biotechnol. Biochem. 1992; 56 (1368312): 355-35610.1271/bbb.56.355Crossref PubMed Scopus (115) Google Scholar, 10An C. Kuda T. Yazaki T. Takahashi H. Kimura B. Flx pyrosequencing analysis of the effects of the brown-algal fermentable polysaccharides alginate and laminaran on rat cecal microbiotas.Appl. Environ. Microbiol. 2013; 79 (23183985): 860-86610.1128/AEM.02354-12Crossref PubMed Scopus (51) Google Scholar, 11Bai S. Chen H. Zhu L. Liu W. Yu H.D. Wang X. Yin Y. Comparative study on the in vitro effects of Pseudomonas aeruginosa and seaweed alginates on human gut microbiota.PLoS ONE. 2017; 12 (28170428): e017157610.1371/journal.pone.0171576Crossref PubMed Scopus (25) Google Scholar, 12Li M. Li G. Shang Q. Chen X. Liu W. Pi X. Zhu L. Yin Y. Yu G. Wang X. In vitro fermentation of alginate and its derivatives by human gut microbiota.Anaerobe. 2016; 39 (26891629): 19-2510.1016/j.anaerobe.2016.02.003Crossref PubMed Scopus (62) Google Scholar, 13Li M. Shang Q. Li G. Wang X. Yu G. Degradation of marine algae-derived carbohydrates by bacteroidetes isolated from human gut microbiota.Mar. Drugs. 2017; 15 (28338633): E9210.3390/md15040092Crossref PubMed Scopus (54) Google Scholar). The population of Bacteroidetes, Bifidobacteria, and Lactobacilli increased in the gut of rats fed alginate (10An C. Kuda T. Yazaki T. Takahashi H. Kimura B. Flx pyrosequencing analysis of the effects of the brown-algal fermentable polysaccharides alginate and laminaran on rat cecal microbiotas.Appl. Environ. Microbiol. 2013; 79 (23183985): 860-86610.1128/AEM.02354-12Crossref PubMed Scopus (51) Google Scholar), and alginate oligosaccharides were bifidogenic in skim milk media (9Akiyama H. Endo T. Nakakita R. Murata K. Yonemoto Y. Okayama K. Effect of depolymerized alginates on the growth of bifidobacteria.Biosci. Biotechnol. Biochem. 1992; 56 (1368312): 355-35610.1271/bbb.56.355Crossref PubMed Scopus (115) Google Scholar). Little is known, however, at the molecular level on alginate breakdown and utilization in the gut beyond the demonstrated substrate specificity of a PL17 enzyme from Bacteriodes eggerthii, found to be polyM-specific (14Mathieu S. Touvrey-Loiodice M. Poulet L. Drouillard S. Vincentelli R. Henrissat B. Skjåk-Bræk G. Helbert W. Ancient acquisition of “alginate utilization loci” by human gut microbiota.Sci. Rep. 2018; 8 (29795267): 807510.1038/s41598-018-26104-1Crossref PubMed Scopus (29) Google Scholar). By contrast, several polysaccharide lyases (PLs) involved in alginate utilization have been described from marine bacteria, including bacteria of the Bacteroidetes phylum (15Kabisch A. Otto A. König S. Becher D. Albrecht D. Schüler M. Teeling H. Amann R.I. Schweder T. Functional characterization of polysaccharide utilization loci in the marine Bacteroidetes 'Gramella forsetii' KT0803.ISME J. 2014; 8 (24522261): 1492-150210.1038/ismej.2014.4Crossref PubMed Scopus (135) Google Scholar, 16Inoue A. Anraku M. Nakagawa S. Ojima T. Discovery of a novel alginate lyase from Nitratiruptor sp. SB155-2 thriving at deep-sea hydrothermal vents and identification of the residues responsible for its heat stability.J. Biol. Chem. 2016; 291 (27231344): 15551-1556310.1074/jbc.M115.713230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 17Kawamoto H. Horibe A. Miki Y. Kimura T. Tanaka K. Nakagawa T. Kawamukai M. Matsuda H. Cloning and sequencing analysis of alginate lyase genes from the marine bacterium Vibrio sp. O2.Mar. Biotechnol. 2006; 8 (16810458): 481-49010.1007/s10126-005-6157-zCrossref PubMed Scopus (46) Google Scholar). PLs are categorized in 37 families in the CAZy database (www.cazy.org) 3Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site. (3Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42 (24270786): D490-D49510.1093/nar/gkt1178Crossref PubMed Scopus (4114) Google Scholar), 10 of which (PL5–7, -14, -15, -17, -18, -32, -34, and -36) contain alginate lyases (18Helbert W. Poulet L. Drouillard S. Mathieu S. Loiodice M. Couturier M. Lombard V. Terrapon N. Turchetto J. Vincentelli R. Henrissat B. Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (30850540): 6063-606810.1073/pnas.1815791116Crossref PubMed Scopus (101) Google Scholar, 19Lombard V. Bernard T. Rancurel C. Brumer H. Coutinho P.M. Henrissat B. A hierarchical classification of polysaccharide lyases for glycogenomics.Biochem. J. 2010; 432 (20925655): 437-44410.1042/BJ20101185Crossref PubMed Scopus (216) Google Scholar). Alginate lyases break the O–C4 bond to uronic acid residues through a β-elimination reaction that leads to formation of the 4,5-unsaturated sugar 4-deoxy-l-erythro-hex-4-enopyranosyluronic acid (denoted as Δ) at the nonreducing end of the released product. Alginate lyases are either endo-acting (2Aarstad O.A. Tøndervik A. Sletta H. Skjåk-Bræk G. Alginate sequencing: an analysis of block distribution in alginates using specific alginate degrading enzymes.Biomacromolecules. 2012; 13 (22148348): 106-11610.1021/bm2013026Crossref PubMed Scopus (86) Google Scholar), initially releasing oligosaccharides that can undergo further degradation, typically to di- and trisaccharides (14Mathieu S. Touvrey-Loiodice M. Poulet L. Drouillard S. Vincentelli R. Henrissat B. Skjåk-Bræk G. Helbert W. Ancient acquisition of “alginate utilization loci” by human gut microbiota.Sci. Rep. 2018; 8 (29795267): 807510.1038/s41598-018-26104-1Crossref PubMed Scopus (29) Google Scholar, 20Mathieu S. Henrissat B. Labre F. Skjåk-Braek G. Helbert W. Functional exploration of the polysaccharide lyase family PL6.PLoS ONE. 2016; 11 (27438604): e015941510.1371/journal.pone.0159415Crossref PubMed Scopus (23) Google Scholar), or exo-acting producing the unsaturated monosaccharide Δ (Fig. 1B) (21Garron M.L. Cygler M. Structural and mechanistic classification of uronic acid-containing polysaccharide lyases.Glycobiology. 2010; 20 (20805221): 1547-157310.1093/glycob/cwq122Crossref PubMed Scopus (179) Google Scholar, 22Xu F. Wang P. Zhang Y.Z. Chen X.L. Diversity of three-dimensional structures and catalytic mechanisms of alginate lyases.Appl. Environ. Microbiol. 2018; 84 (29150496): e02017-e0204010.1128/AEM.02040-17Crossref Scopus (54) Google Scholar). A PL6 family enzyme has yet to be characterized from the gut niche. PL6 is multispecific and can be divided into three subfamilies (19Lombard V. Bernard T. Rancurel C. Brumer H. Coutinho P.M. Henrissat B. A hierarchical classification of polysaccharide lyases for glycogenomics.Biochem. J. 2010; 432 (20925655): 437-44410.1042/BJ20101185Crossref PubMed Scopus (216) Google Scholar), PL6_1 of endo- and exo-acting alginate or dermatan sulfate–specific enzymes, and PL6_2 and PL6_3, which are reported to contain only polyMG endolyases (20Mathieu S. Henrissat B. Labre F. Skjåk-Braek G. Helbert W. Functional exploration of the polysaccharide lyase family PL6.PLoS ONE. 2016; 11 (27438604): e015941510.1371/journal.pone.0159415Crossref PubMed Scopus (23) Google Scholar). Most characterized alginate lyases of PL6_1 have broad substrate specificity on polyMG and polyG (20Mathieu S. Henrissat B. Labre F. Skjåk-Braek G. Helbert W. Functional exploration of the polysaccharide lyase family PL6.PLoS ONE. 2016; 11 (27438604): e015941510.1371/journal.pone.0159415Crossref PubMed Scopus (23) Google Scholar, 23Lee S.I. Choi S.H. Lee E.Y. Kim H.S. Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2.Appl. Microbiol. Biotechnol. 2012; 95 (22805784): 1643-165310.1007/s00253-012-4266-yCrossref PubMed Scopus (59) Google Scholar, 24Li S. Wang L. Han F. Gong Q. Yu W. Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp. Kz7.J. Biochem. 2016; 159 (26232404): 77-8610.1093/jb/mvv076Crossref PubMed Scopus (46) Google Scholar), but a few, e.g. Patl3640 and Pedsa0631 from Pseudoalteromonas atlantica and Pseudobacter saltans respectively, are strictly polyG-specific (20Mathieu S. Henrissat B. Labre F. Skjåk-Braek G. Helbert W. Functional exploration of the polysaccharide lyase family PL6.PLoS ONE. 2016; 11 (27438604): e015941510.1371/journal.pone.0159415Crossref PubMed Scopus (23) Google Scholar). With regard to three-dimensional structures, alginate lyases adopt several different folds: β-jelly roll; (α/α)n toroid; and parallel β-helix, and some are multimodular (21Garron M.L. Cygler M. Structural and mechanistic classification of uronic acid-containing polysaccharide lyases.Glycobiology. 2010; 20 (20805221): 1547-157310.1093/glycob/cwq122Crossref PubMed Scopus (179) Google Scholar, 22Xu F. Wang P. Zhang Y.Z. Chen X.L. Diversity of three-dimensional structures and catalytic mechanisms of alginate lyases.Appl. Environ. Microbiol. 2018; 84 (29150496): e02017-e0204010.1128/AEM.02040-17Crossref Scopus (54) Google Scholar, 25Garron M.L. Cygler M. Uronic polysaccharide degrading enzymes.Curr. Opin. Struct. Biol. 2014; 28 (25156747): 87-9510.1016/j.sbi.2014.07.012Crossref PubMed Scopus (37) Google Scholar). PL6 displays a right-handed parallel β-helix fold similar to several other polysaccharide lyase families (21Garron M.L. Cygler M. Structural and mechanistic classification of uronic acid-containing polysaccharide lyases.Glycobiology. 2010; 20 (20805221): 1547-157310.1093/glycob/cwq122Crossref PubMed Scopus (179) Google Scholar, 22Xu F. Wang P. Zhang Y.Z. Chen X.L. Diversity of three-dimensional structures and catalytic mechanisms of alginate lyases.Appl. Environ. Microbiol. 2018; 84 (29150496): e02017-e0204010.1128/AEM.02040-17Crossref Scopus (54) Google Scholar, 25Garron M.L. Cygler M. Uronic polysaccharide degrading enzymes.Curr. Opin. Struct. Biol. 2014; 28 (25156747): 87-9510.1016/j.sbi.2014.07.012Crossref PubMed Scopus (37) Google Scholar). The first PL6 crystal structure was determined for the single domain chondroitin B lyase from Pedobacter heparinus DSM 2366 (PBD code 1OFL) that degrades dermatan sulfate (26Huang W. Matte A. Li Y. Kim Y.S. Linhardt R.J. Su H. Cygler M. Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 Å resolution.J. Mol. Biol. 1999; 294 (10600383): 1257-126910.1006/jmbi.1999.3292Crossref PubMed Scopus (97) Google Scholar). Recently, structures also became available for two marine bacterial alginate lyases, namely the polyG-specific homodimeric, two-domain exolyase AlyGC from Paraglaciecola chatamensis S18K6T (PDB code 5GKQ) that produces Δ, and the monomeric, single-domain endolyase AlyF from Vibrio splendidus OU2 (PDB code 5Z9T), releasing unsaturated trisaccharides from alginates and polyG (27Xu F. Dong F. Wang P. Cao H.Y. Li C.Y. Li P.Y. Pang X.H. Zhang Y.Z. Chen X.L. Novel molecular insights into the catalytic mechanism of marine bacterial alginate lyase AlyGC from polysaccharide lyase Family 6.J. Biol. Chem. 2017; 292 (28154171): 4457-446810.1074/jbc.M116.766030Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 28Lyu Q. Zhang K. Shi Y. Li W. Diao X. Liu W. Structural insights into a novel Ca2+-independent PL-6 alginate lyase from Vibrio OU02 identify the possible subsites responsible for product distribution.Biochim. Biophys. Acta. 2019; 1863 (31004719): 1167-117610.1016/j.bbagen.2019.04.013Crossref Scopus (28) Google Scholar). PL6 thus encompasses various types of specificity toward alginates as well as for dermatan sulfate, an O-sulfated glycosaminoglycan of alternating 1,3-β-d-galactosamine and 1,4 α-l-iduronic acid (20Mathieu S. Henrissat B. Labre F. Skjåk-Braek G. Helbert W. Functional exploration of the polysaccharide lyase family PL6.PLoS ONE. 2016; 11 (27438604): e015941510.1371/journal.pone.0159415Crossref PubMed Scopus (23) Google Scholar, 21Garron M.L. Cygler M. Structural and mechanistic classification of uronic acid-containing polysaccharide lyases.Glycobiology. 2010; 20 (20805221): 1547-157310.1093/glycob/cwq122Crossref PubMed Scopus (179) Google Scholar). PL6 is proposed to have conserved lysine and arginine residues acting as catalytic residues. This is opposed to alginate lyases of other PL families in which tyrosine and histidine are identified as catalytic residues (21Garron M.L. Cygler M. Structural and mechanistic classification of uronic acid-containing polysaccharide lyases.Glycobiology. 2010; 20 (20805221): 1547-157310.1093/glycob/cwq122Crossref PubMed Scopus (179) Google Scholar, 22Xu F. Wang P. Zhang Y.Z. Chen X.L. Diversity of three-dimensional structures and catalytic mechanisms of alginate lyases.Appl. Environ. Microbiol. 2018; 84 (29150496): e02017-e0204010.1128/AEM.02040-17Crossref Scopus (54) Google Scholar, 25Garron M.L. Cygler M. Uronic polysaccharide degrading enzymes.Curr. Opin. Struct. Biol. 2014; 28 (25156747): 87-9510.1016/j.sbi.2014.07.012Crossref PubMed Scopus (37) Google Scholar). In PL6, the negatively charged C6 carboxyl group accommodated at subsite +1 is neutralized by Ca2+. This reduces the pKa of the C5 proton facilitating its abstraction by the general base catalyst (Fig. 1B) (26Huang W. Matte A. Li Y. Kim Y.S. Linhardt R.J. Su H. Cygler M. Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 Å resolution.J. Mol. Biol. 1999; 294 (10600383): 1257-126910.1006/jmbi.1999.3292Crossref PubMed Scopus (97) Google Scholar, 27Xu F. Dong F. Wang P. Cao H.Y. Li C.Y. Li P.Y. Pang X.H. Zhang Y.Z. Chen X.L. Novel molecular insights into the catalytic mechanism of marine bacterial alginate lyase AlyGC from polysaccharide lyase Family 6.J. Biol. Chem. 2017; 292 (28154171): 4457-446810.1074/jbc.M116.766030Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 29Michel G. Pojasek K. Li Y. Sulea T. Linhardt R.J. Raman R. Prabhakar V. Sasisekharan R. Cygler M. The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent catalytic machinery.J. Biol. Chem. 2004; 279 (15155751): 32882-3289610.1074/jbc.M403421200Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 30Gacesa P. Alginate-modifying enzymes. A proposed unified mechanism of action for the lyases and epimerases.FEBS Lett. 1987; 212: 199-20210.1016/0014-5793(87)81344-3Crossref Scopus (164) Google Scholar). However, a calcium-independent PL6 alginate lyase was reported recently (28Lyu Q. Zhang K. Shi Y. Li W. Diao X. Liu W. Structural insights into a novel Ca2+-independent PL-6 alginate lyase from Vibrio OU02 identify the possible subsites responsible for product distribution.Biochim. Biophys. Acta. 2019; 1863 (31004719): 1167-117610.1016/j.bbagen.2019.04.013Crossref Scopus (28) Google Scholar). The proton abstraction can occur either in syn configuration, having the C5 proton and the glycosidic oxygen of the bond to be cleaved situated on the same side of the sugar ring in the transition state, as is the case of M-specific lyases, or in anti configuration when these groups are placed on opposite sides of the sugar ring, as for breaking G-linkages (21Garron M.L. Cygler M. Structural and mechanistic classification of uronic acid-containing polysaccharide lyases.Glycobiology. 2010; 20 (20805221): 1547-157310.1093/glycob/cwq122Crossref PubMed Scopus (179) Google Scholar, 22Xu F. Wang P. Zhang Y.Z. Chen X.L. Diversity of three-dimensional structures and catalytic mechanisms of alginate lyases.Appl. Environ. Microbiol. 2018; 84 (29150496): e02017-e0204010.1128/AEM.02040-17Crossref Scopus (54) Google Scholar). The majority of characterized PL6 members produce di- and tetrasaccharides as end products (20Mathieu S. Henrissat B. Labre F. Skjåk-Braek G. Helbert W. Functional exploration of the polysaccharide lyase family PL6.PLoS ONE. 2016; 11 (27438604): e015941510.1371/journal.pone.0159415Crossref PubMed Scopus (23) Google Scholar). Here, we show that BcelPL6 of PL6 subfamily 1 from the human commensal gut bacterium Bacteroides cellulosilyticus CRE21 is a monomeric, single-domain polyM-specific enzyme. The crystal structure is solved to 1.3 Å resolution and contains a long, highly-conserved asparagine ladder. The residues at the active site provide insights into specificity determinants in PL6. Most known genes from marine Bacteroides associated with alginate utilization, except from PL7, have orthologues in B. cellulosilyticus CRE21 as identified by a BLAST search. Searching against nonredundant protein sequences revealed that BcelPL6 is conserved in Bacteroides with homologues of >85% sequence identity in strains of human gut Bacteroides intestinalis, Bacteroides sp. 14(A), Bacteroides oleiciplenus, Bacteroides timonensis, and Bacteroides stercorirosoris. Although gene up-regulation has not been analyzed for human gut Bacteroides growing on alginate, it has been reported in the cases of several members of PL6, PL7, and PL17 from the marine Gramella forsetti that belongs to the Bacteroidetes phylum and for Alteromonas macleodii (15Kabisch A. Otto A. König S. Becher D. Albrecht D. Schüler M. Teeling H. Amann R.I. Schweder T. Functional characterization of polysaccharide utilization loci in the marine Bacteroidetes 'Gramella forsetii' KT0803.ISME J. 2014; 8 (24522261): 1492-150210.1038/ismej.2014.4Crossref PubMed Scopus (135) Google Scholar, 31Koch H. Dürwald A. Schweder T. Noriega-Ortega B. Vidal-Melgosa S. Hehemann J.H. Dittmar T. Freese H.M. Becher D. Simon M. Wietz M. Biphasic cellular adaptations and ecological implications of Alteromonas macleodii degrading a mixture of algal polysaccharides.ISME J. 2019; 13 (30116038): 92-10310.1038/s41396-018-0252-4Crossref PubMed Scopus (37) Google Scholar). B. cellulosilyticus of the HGM encodes polysaccharide utilization loci (PULs) involved in degradation and product uptake of polysaccharides, e.g. starch (32Terrapon N. Lombard V. Drula É. Lapébie P. Al-Masaudi S. Gilbert H.J. Henrissat B. PULDB: the expanded database of polysaccharide utilization loci.Nucleic Acids Res. 2018; 46 (29088389): D677-D68310.1093/nar/gkx1022Crossref PubMed Scopus (113) Google Scholar). BcelPL6 was not annotated to a PUL (32Terrapon N. Lombard V. Drula É. Lapébie P. Al-Masaudi S. Gilbert H.J. Henrissat B. PULDB: the expanded database of polysaccharide utilization loci.Nucleic Acids Res. 2018; 46 (29088389): D677-D68310.1093/nar/gkx1022Crossref PubMed Scopus (113) Google Scholar), but BcelPL6 orthologues are predicted along with an annotated PL17 in PULs of B. intestinalis DSM 17393, B. ovatus NLAE-zl-H73, and B. xylanisolvens NLAE-zl-G339 of the HGM (14Mathieu S. Touvrey-Loiodice M. Poulet L. Drouillard S. Vincentelli R. Henrissat B. Skjåk-Bræk G. Helbert W. Ancient acquisition of “alginate utilization loci” by human gut microbiota.Sci. Rep. 2018; 8 (29795267): 807510.1038/s41598-018-26104-1Crossref PubMed Scopus (29) Google Scholar, 32Terrapon N. Lombard V. Drula É. Lapébie P. Al-Masaudi S. Gilbert H.J. Henrissat B. PULDB: the expanded database of polysaccharide utilization loci.Nucleic Acids Res. 2018; 46 (29088389): D677-D68310.1093/nar/gkx1022Crossref PubMed Scopus (113) Google Scholar). A Pfam domain search suggested BcelPL6 is a chondroitinase B, yet another PL6 specificity. This reflects that target substrate variation probably correlates with subtle changes in the active-site structure in PL6 (26Huang W. Matte A. Li Y. Kim Y.S. Linhardt R.J. Su H. Cygler M. Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 Å resolution.J. Mol. Biol. 1999; 294 (10600383): 1257-126910.1006/jmbi.1999.3292Crossref PubMed Scopus (97) Google Scholar, 27Xu F. Dong F. Wang P. Cao H.Y. Li C.Y. Li P.Y. Pang X.H. Zhang Y.Z. Chen X.L. Novel molecular insights into the catalytic mechanism of marine bacterial alginate lyase AlyGC from polysaccharide lyase Family 6.J. Biol. Chem. 2017; 292 (28154171): 4457-446810.1074/jbc.M116.766030Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 28Lyu Q. Zhang K. Shi Y. Li W. Diao X. Liu W. Structural insights into a novel Ca2+-independent PL-6 alginate lyase from Vibrio OU02 identify the possible subsites responsible for product distribution.Biochim. Biophys. Acta. 2019; 1863 (31004719): 1167-117610.1016/j.bbagen.2019.04.013Crossref Scopus (28) Google Scholar, 29Michel G. Pojasek K. Li Y. Sulea T. Linhardt R.J. Raman R. Prabhakar V. Sasisekharan R. Cygler M. The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent catalytic machinery.J. Biol. Chem. 2004; 279 (15155751): 32882-3289610.1074/jbc.M403421200Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Therefore, sequence-based prediction of PL6 specificities is currently not reliable. BcelPL6 catalyzed the release of products with unsaturated nonreducing ends (Fig. 1B) from alginate (Fig. 2A) and polyM (Fig. 2B). The reactions followed Michaelis-Menten kinetics, and kcat was 8-fold higher for polyM (43.4 ± 1.6 s−1) than alginate (kcat = 5.4 ± 0.15 s−1), whereas Km was 3-fold lower for alginate (0.59 ± 0.04 mg ml−1) than polyM (Km = 1.96 ± 0.18 mg ml−1) (Table 1). Activity was barely detected toward polyG (Fig. 2C; Table 1; Fig. S2A) and polyMG (Table 1; Fig. S2B) even at high concentrations (6 μm) of BcelPL6, and the observed very low rates of degradation of 0–2.0 mg ml−1 polyG or polyMG did not follow Michaelis-Menten kinetics (Table 1). Trace of product formation from polyG possibly stems from the 3% M being found in the used polyG candidate substrate. Moreover, BcelPL6 did not degrade acetylated polyM that mimics bacterial alginate (Table 1 and" @default.
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• W2974863485 date "2019-11-01" @default.
• W2974863485 modified "2023-10-16" @default.
• W2974863485 title "Structural and functional aspects of mannuronic acid–specific PL6 alginate lyase from the human gut microbe Bacteroides cellulosilyticus" @default.
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• W2974863485 doi "https://doi.org/10.1074/jbc.ra119.010206" @default.
• W2974863485 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6879350" @default.
• W2974863485 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/31530640" @default.
• W2974863485 hasPublicationYear "2019" @default.

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