Crystal structure of a neoagarobiose-producing GH16 family β-agarase from Persicobacter sp. CCB-QB2

  • Aik-Hong TehEmail author
  • Nur Hafizah Fazli
  • Go Furusawa
Biotechnologically relevant enzymes and proteins


PdAgaC from the marine bacterium Persicobacter sp. CCB-QB2 is a β-agarase belonging to the glycoside hydrolase family 16 (GH16). It is one of only a handful of endo-acting GH16 β-agarases able to degrade agar completely to produce neoagarobiose (NA2). The crystal structure of PdAgaC’s catalytic domain, which has one of the highest Vmax value at 2.9 × 103 U/mg, was determined in order to understand its unique mechanism. The catalytic domain is made up of a typical β-jelly roll fold with two additional insertions, and a well-conserved but wider substrate-binding cleft with some minor changes. Among the unique differences, two unconserved residues, Asn226 and Arg286, may potentially contribute additional hydrogen bonds to subsites −1 and +2, respectively, while a third, His185 from one of the additional insertions, may further contribute another bond to subsite +2. These additional hydrogen bonds may probably have enhanced PdAgaC’s affinity for short agaro-oligosaccharides such as neoagarotetraose (NA4), rendering it capable of binding NA4 strongly enough for rapid degradation into NA2.


β-agarase Crystal structure Substrate binding Neoagarobiose Persicobacter 


Funding information

This study was funded by the Mangrove Microbial Chemical Biology grant from Universiti Sains Malaysia (1001/PCCB/870009).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_10237_MOESM1_ESM.pdf (119 kb)
ESM 1 (PDF 119 kb)


  1. Allouch J, Jam M, Helbert W, Barbeyron T, Kloareg B, Henrissat B, Czjzek M (2003) The three-dimensional structures of two β-agarases. J Biol Chem 278:47171–47180. CrossRefPubMedGoogle Scholar
  2. Allouch J, Helbert W, Henrissat B, Czjzek M (2004) Parallel substrate binding sites in a β-agarase suggest a novel mode of action on double-helical agarose. Structure 12:623–632. CrossRefPubMedGoogle Scholar
  3. An K, Shi X, Cui F, Cheng J, Liu N, Zhao X, Zhang XH (2018) Characterization and overexpression of a glycosyl hydrolase family 16 beta-agarase YM01-1 from marine bacterium Catenovulum agarivorans YM01T. Protein Expr Purif 143:1–8. CrossRefPubMedGoogle Scholar
  4. Boratyn GM, Schaffer AA, Agarwala R, Altschul SF, Lipman DJ, Madden TL (2012) Domain enhanced lookup time accelerated BLAST. Biol Direct 7:12. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chi WJ, Park DY, Seo YB, Chang YK, Lee SY, Hong SK (2014) Cloning, expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1. Appl Microbiol Biotechnol 98:4545–4555. CrossRefPubMedGoogle Scholar
  6. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Furusawa G, Lau NS, Suganthi A, Amirul AA (2017) Agarolytic bacterium Persicobacter sp. CCB-QB2 exhibited a diauxic growth involving galactose utilization pathway. Microbiologyopen 6. CrossRefGoogle Scholar
  8. Hafizah NF, Teh AH, Furusawa G (2019) Biochemical characterization of thermostable and detergent-tolerant β-agarase, PdAgaC, from Persicobacter sp. CCB-QB2. Appl Biochem Biotechnol 187:770–781. CrossRefPubMedGoogle Scholar
  9. Hehemann JH, Correc G, Barbeyron T, Helbert W, Czjzek M, Michel G (2010) Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature 464:908–912. CrossRefPubMedGoogle Scholar
  10. Hehemann JH, Correc G, Thomas F, Bernard T, Barbeyron T, Jam M, Helbert W, Michel G, Czjzek M (2012) Biochemical and structural characterization of the complex agarolytic enzyme system from the marine bacterium Zobellia galactanivorans. J Biol Chem 287:30571–30584. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Henshaw J, Horne-Bitschy A, van Bueren AL, Money VA, Bolam DN, Czjzek M, Ekborg NA, Weiner RM, Hutcheson SW, Davies GJ, Boraston AB, Gilbert HJ (2006) Family 6 carbohydrate binding modules in β-agarases display exquisite selectivity for the non-reducing termini of agarose chains. J Biol Chem 281:17099–17107. CrossRefPubMedGoogle Scholar
  12. Holm L, Laakso LM (2016) Dali server update. Nucleic Acids Res 44:W351–W355. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Kim J, Hong SK (2012) Isolation and characterization of an agarase-producing bacterial strain, Alteromonas sp. GNUM-1, from the West Sea, Korea. J Microbiol Biotechnol 22:1621–1628CrossRefGoogle Scholar
  14. Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Labourel A, Jam M, Jeudy A, Hehemann JH, Czjzek M, Michel G (2014) The β-glucanase ZgLamA from Zobellia galactanivorans evolved a bent active site adapted for efficient degradation of algal laminarin. J Biol Chem 289:2027–2042. CrossRefPubMedGoogle Scholar
  16. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495. CrossRefGoogle Scholar
  17. Matard-Mann M, Bernard T, Leroux C, Barbeyron T, Larocque R, Prechoux A, Jeudy A, Jam M, Nyvall Collen P, Michel G, Czjzek M (2017) Structural insights into marine carbohydrate degradation by family GH16 κ-carrageenases. J Biol Chem 292:19919–19934. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53:240–255. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Naretto A, Fanuel M, Ropartz D, Rogniaux H, Larocque R, Czjzek M, Tellier C, Michel G (2019) The agar-specific hydrolase ZgAgaC from the marine bacterium Zobellia galactanivorans defines a new GH16 protein subfamily. J Biol Chem 294:6923–6939. CrossRefPubMedGoogle Scholar
  20. Park DY, Chi WJ, Park JS, Chang YK, Hong SK (2015) Cloning, expression, and biochemical characterization of a GH16 β-agarase AgaH71 from Pseudoalteromonas hodoensis H7. Appl Biochem Biotechnol 175:733–747. CrossRefPubMedGoogle Scholar
  21. Pluvinage B, Grondin JM, Amundsen C, Klassen L, Moote PE, Xiao Y, Thomas D, Pudlo NA, Anele A, Martens EC, Inglis GD, Uwiera RER, Boraston AB, Abbott DW (2018) Molecular basis of an agarose metabolic pathway acquired by a human intestinal symbiont. Nat Commun 9:1043. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42:W320–W324. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Rozewicki J, Li S, Amada KM, Standley DM, Katoh K (2019) MAFFT-DASH: integrated protein sequence and structural alignment. Nucleic Acids Res 47:W5–W10. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Sehnal D, Svobodova Varekova R, Berka K, Pravda L, Navratilova V, Banas P, Ionescu CM, Otyepka M, Koca J (2013) MOLE 2.0: advanced approach for analysis of biomacromolecular channels. Aust J Chem 5:39. CrossRefGoogle Scholar
  26. Sim PF, Furusawa G, Teh AH (2017) Functional and structural studies of a multidomain alginate lyase from Persicobacter sp. CCB-QB2. Sci Rep 7:13656.,288-1 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Takagi E, Hatada Y, Akita M, Ohta Y, Yokoi G, Miyazaki T, Nishikawa A, Tonozuka T (2015) Crystal structure of the catalytic domain of a GH16 beta-agarase from a deep-sea bacterium, Microbulbifer thermotolerans JAMB-A94. Biosci Biotechnol Biochem 79:625–632. CrossRefPubMedGoogle Scholar
  28. Teh AH, Saito JA, Baharuddin A, Tuckerman JR, Newhouse JS, Kanbe M, Newhouse EI, Rahim RA, Favier F, Didierjean C, Sousa EH, Stott MB, Dunfield PF, Gonzalez G, Gilles-Gonzalez MA, Najimudin N, Alam M (2011) Hell’s Gate globin I: an acid and thermostable bacterial hemoglobin resembling mammalian neuroglobin. FEBS Lett 585:3250–3258. CrossRefPubMedGoogle Scholar
  29. Thomas F, Bordron P, Eveillard D, Michel G (2017) Gene expression analysis of Zobellia galactanivorans during the degradation of algal polysaccharides reveals both substrate-specific and shared transcriptome-wide responses. Front Microbiol 8:1808. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Vagin A, Teplyakov A (2010) Molecular replacement with MOLREP. Acta Crystallogr D Biol Crystallogr 66:22–25. CrossRefPubMedGoogle Scholar
  31. Yan S, Yu M, Wang Y, Shen C, Zhang XH (2011) Catenovulum agarivorans gen. nov., sp. nov., a peritrichously flagellated, chain-forming, agar-hydrolysing gammaproteobacterium from seawater. Int J Syst Evol Microbiol 61:2866–2873. CrossRefPubMedGoogle Scholar
  32. Yang J, Kloek AP, Goldberg DE, Mathews FS (1995) The structure of Ascaris hemoglobin domain I at 2.2 A resolution: molecular features of oxygen avidity. Proc Natl Acad Sci U S A 92:4224–4228CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Centre for Chemical BiologyUniversiti Sains MalaysiaPenangMalaysia

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