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New Family of Ulvan Lyases Identified in Three Isolates from the Alteromonadales Order*

  • Moran Kopel
    Affiliations
    From the Institute for Nanotechnology and Advanced Materials, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, 52900 Ramat Gan, Israel and
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  • William Helbert
    Correspondence
    To whom correspondence may be addressed. Tel.: 33-4-7603-7661; Fax: 33-4-7654-7203;
    Affiliations
    the Centre de efRecherches sur les Macromolécules Végétales (UPR-CNRS 5301), Université Joseph Fourier and Institut de Chimie Moléculaire de Grenoble (ICMG, FR-CNRS 2607), Grenoble cedex 9, France
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  • Yana Belnik
    Affiliations
    From the Institute for Nanotechnology and Advanced Materials, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, 52900 Ramat Gan, Israel and
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  • Vitaliy Buravenkov
    Affiliations
    From the Institute for Nanotechnology and Advanced Materials, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, 52900 Ramat Gan, Israel and
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  • Asael Herman
    Affiliations
    From the Institute for Nanotechnology and Advanced Materials, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, 52900 Ramat Gan, Israel and
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  • Ehud Banin
    Correspondence
    To whom correspondence may be addressed. Tel.: 972-3-531-7288; Fax: 972-3-738-4511;
    Affiliations
    From the Institute for Nanotechnology and Advanced Materials, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, 52900 Ramat Gan, Israel and
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  • Author Footnotes
    * This work was supported in part by an infrastructure grant from the Israel Ministry of Science, Technology and Space (to E. B.) and by a Ph. D. fellowship (to M. K.). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:January 13, 2016DOI:https://doi.org/10.1074/jbc.M115.673947
      Ulvan is the main polysaccharide component of the Ulvales (green seaweed) cell wall. It is composed of disaccharide building blocks comprising 3-sulfated rhamnose linked to d-glucuronic acid (GlcUA), l-iduronic acid (IdoUA), or d-xylose (Xyl). The degradation of ulvan requires ulvan lyase, which catalyzes the endolytic cleavage of the glycoside bond between 3-sulfated rhamnose and uronic acid according to a β-elimination mechanism. The first characterized ulvan lyase was identified in Nonlabens ulvanivorans, an ulvanolytic bacterial isolate. In the current study, we have identified and biochemically characterized novel ulvan lyases from three Alteromonadales isolated bacteria. Two homologous ulvan lyases (long and short) were found in each of the bacterial genomes. The protein sequences have no homology to the previously reported ulvan lyases and therefore are the first representatives of a new family of polysaccharide lyases. The enzymes were heterologously expressed in Escherichia coli to determine their mode of action. The heterologous expressed enzymes were secreted into the milieu subsequent to their signal sequence cleavage. An endolytic mode of action was observed and studied using gel permeation chromatography and 1H NMR. In contrast to N. ulvanivorans ulvan lyase, cleavage occurred specifically at the GlcUA residues. In light of the genomic context and modular structure of the ulvan lyase families identified to date, we propose that two ulvan degradation pathways evolved independently.

      Introduction

      Ulvales are green algae that proliferate in eutrophicated coastal waters. Members of this family are commonly grown and collected for food or feed, although currently most of the biomass is put to limited use (
      • Lahaye M.
      • Brunel M.
      • Bonnin E.
      Fine chemical structure analysis of oligosaccharides produced by an ulvan-lyase degradation of the water-soluble cell-wall polysaccharides from Ulva sp, (Ulvales, Chlorophyta).
      ,
      • Lahaye M.
      • Robic A.
      Structure and functional properties of ulvan, a polysaccharide from green seaweeds.
      ). Among the polymers synthesized by these algae, ulvan is the most abundant polysaccharide in the architecture of the cell wall (
      • Lahaye M.
      • Robic A.
      Structure and functional properties of ulvan, a polysaccharide from green seaweeds.
      ). This complex water-soluble anionic polysaccharide represents up to 29% of the algae dry weight (
      • Robic A.
      • Gaillard C.
      • Sassi J.-F.
      • Lerat Y.
      • Lahaye M.
      Ultrastructure of ulvan: a polysaccharide from green seaweeds.
      ). Although the constituent ratio might vary according to algae growth conditions, it is mainly composed of 3-sulfated rhamnose (R3S),
      The abbreviations used are: R3S, 3-sulfated rhamnose; GlcUA, d-glucuronic acid; IdoUA, l-iduronic acid; Xyl, d-xylose; CM, conditioned medium; GH, glycoside hydrolase.
      d-glucuronic acid (GlcUA), l-iduronic acid (IdoUA), and d-xylose (Xyl) (
      • Lahaye M.
      • Robic A.
      Structure and functional properties of ulvan, a polysaccharide from green seaweeds.
      ). Ulvan building blocks appear frequently as repeating disaccharides comprising either GlcUA or IdoUA linked to R3S, termed ulvanobiuronic acid A or B, respectively (
      • Lahaye M.
      • Robic A.
      Structure and functional properties of ulvan, a polysaccharide from green seaweeds.
      ). In addition, disaccharide moieties made of R3S linked to Xyl occur in lower amounts.
      Ulvan's unique chemical and physicochemical properties make it an attractive candidate for several applications in the food/feed, agriculture, pharmaceutical, and biomaterials industries (
      • Lahaye M.
      • Robic A.
      Structure and functional properties of ulvan, a polysaccharide from green seaweeds.
      ). In contrast to polysaccharides extracted from brown and red algae (e.g. alginate and agar, respectively), which are widely used in industry for their gelling and thickening properties, polysaccharides of green algae are less exploited. Expanding our understanding of ulvan structure and its enzymatic degradation would enable more extensive biomass utilization.
      Thus far, only a few ulvan-degrading enzymes have been isolated from both marine and terrestrial microorganisms. Some of them, like the glucuronan lyases isolated from Sinorhizobium meliloti (
      • Da Costa A.
      • Michaud P.
      • Petit E.
      • Heyraud A.
      • Colin-Morel P.
      • Courtois B.
      • Courtois J.
      Purification and properties of a glucuronan lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472).
      ) and Trichoderma sp. GL2 (
      • Delattre C.
      • Michaud P.
      • Keller C.
      • Elboutachfaiti R.
      • Beven L.
      • Courtois B.
      • Courtois J.
      Purification and characterization of a novel glucuronan lyase from Trichoderma sp. GL2.
      ), possess limited ulvanolytic activity. The first ulvan lyase activity was found in a marine bacterium by Lahaye et al. (
      • Lahaye M.
      • Brunel M.
      • Bonnin E.
      Fine chemical structure analysis of oligosaccharides produced by an ulvan-lyase degradation of the water-soluble cell-wall polysaccharides from Ulva sp, (Ulvales, Chlorophyta).
      ), who employed the newly discovered enzyme extract to degrade ulvan for structural analysis. More recently, several bacterial strains capable of metabolizing ulvan were isolated from the feces of a sea slug, Aplysia punctate, fed with Ulva. In this way, the Gram-negative Nonlabens ulvanivorans PLR was identified (
      • Barbeyron T.
      • Lerat Y.
      • Sassi J.-F.
      • Le Panse S.
      • Helbert W.
      • Collén P.N.
      Persicivirga ulvanivorans sp nov., a marine member of the family Flavobacteriaceae that degrades ulvan from green algae.
      ,
      • Yi H.
      • Chun J.
      Unification of the genera Nonlabens, Persicivirga, Sandarakinotalea and Stenothermobacter into a single emended genus, Nonlabens, and description of Nonlabens agnitussp nov.
      ) and its genome was sequenced (
      • Kopel M.
      • Helbert W.
      • Henrissat B.
      • Doniger T.
      • Banin E.
      Draft genome sequence of Nonlabens ulvanivorans, an ulvan-degrading bacterium.
      ). A novel ulvan lyase was purified from N. ulvanivorans batch culture, sequenced, and heterologously overexpressed in Escherichia coli, and the enzyme's ability to depolymerize ulvan biochemically characterized (
      • Nyvall Collén P.
      • Sassi J.-F.
      • Rogniaux H.
      • Marfaing H.
      • Helbert W.
      Ulvan lyases isolated from the Flavobacteria Persicivirga ulvanivorans are the first members of a new polysaccharide lyase family.
      ). N. ulvanivorans ulvan lyase was reported to cleave ulvan at the β(1→4) glyosidic bond between R3S and GlcUA or IdoUA via the β-elimination mechanism. The proton at the C5 position is abstracted, regardless of its configuration (syn for IdoUA or anti for GlcUA) with the hydroxyl group at C4 (Fig. 1). The β-eliminative cleavage results in the formation of a reducing end on one fragment and an unsaturated ring (Δ, 4-deoxy-l-threo-hex-4-enopyranosiduronic acid) on the non-reducing end of the second fragment (
      • Garron M.-L.
      • Cygler M.
      Structural and mechanistic classification of uronic acid-containing polysaccharide lyases.
      ). Because the protein sequence of N. ulvanivorans ulvan lyase had no characterized homolog in the databases, it was considered the first representative of a new family of polysaccharide lyases.
      Figure thumbnail gr1
      FIGURE 1.Ulvan lyase mode of action. A and B, chemical scheme of N. ulvanivorans ulvan lyase (UL) cleaving ulvan at the β(1→4) glyosidic bond between R3S and GlcUA (A) or IdoUA (B) via β-elimination mechanism. The β-eliminative cleavage results in the formation of a reducing end on one fragment and an unsaturated ring (Δ, 4-deoxy-l-threo-hex-4-enopyranosiduronic acid) on the non-reducing end of the second fragment.
      Sequence encoding an unsaturated β-glucuronyl hydrolase belonging to the glycoside hydrolase family GH105 (based on the Carbohydrate-Active Enzymes (CAZy) database) was found in the vicinity of the ulvan lyase in the N. ulvanivorans genome. This enzyme was shown to cleave specifically the unsaturated non-reducing end of the end products of the ulvan lyase (
      • Collén P.N.
      • Jeudy A.
      • Sassi J.-F.
      • Groisillier A.
      • Czjzek M.
      • Coutinho P.M.
      • Helbert W.
      A novel unsaturated β-glucuronyl hydrolase involved in ulvan degradation unveils the versatility of stereochemistry requirements in family GH105.
      ). The spatial proximity within the genome of these two ulvan-degrading enzymes pointed to occurrence of polysaccharide utilization loci. With this premise in mind, we sequenced the genome of N. ulvanivorans and three additional ulvanolytic Alteromonadales isolates: Alteromonas sp. LOR, Alteromonas sp. LTR, and Pseudoalteromonas sp. PLSV (
      • Kopel M.
      • Helbert W.
      • Henrissat B.
      • Doniger T.
      • Banin E.
      Draft genome sequences of two ulvan-degrading isolates, strains LTR and LOR, that belong to the Alteromonas genus.
      ,
      • Kopel M.
      • Helbert W.
      • Henrissat B.
      • Doniger T.
      • Banin E.
      Draft genome sequence of Pseudoalteromonas sp. strain PLSV, an ulvan-degrading bacterium.
      ). Inspection of the genomes did not reveal genes encoding proteins homologous to N. ulvanivorans ulvan lyase. This led us to hypothesize that additional ulvan lyases that do not share sequence similarity with the known ulvan lyase may be encoded in the genomes of the three other ulvanolytic strains. Here, we report identification and biochemical characterization of four novel ulvan lyases, which belong to a new polysaccharide lyase family now being established
      B. Henrissat, personal communication.
      and are unrelated to N. ulvanivorans ulvan lyase.

      Discussion

      Putative protein-coding genes located in a gene cluster likely involved in carbohydrate metabolism were cloned and overexpressed, and the resulting proteins were screened for ulvanolytic activity. This targeted screening strategy enabled identification of a new ulvan lyase, LOR_107, with a protein sequence unrelated to the previously characterized N. ulvanivorans ulvan lyase (
      • Nyvall Collén P.
      • Sassi J.-F.
      • Rogniaux H.
      • Marfaing H.
      • Helbert W.
      Ulvan lyases isolated from the Flavobacteria Persicivirga ulvanivorans are the first members of a new polysaccharide lyase family.
      ). 1H NMR analysis of the degradation end products, composed essentially of Δ-R3S and Δ-R3S-IdoUA-R3S, indicated that LOR_107 ulvan lyase cleaves specifically the β(1→4) glycoside bond between GlcUA and R3S residues according to a β-elimination mechanism. This substrate specificity is a unique feature, as the previously identified ulvan lyase in N. ulvanivorans cleaves both GlcUA and IdoUA residues by abstraction of the proton on the C5 position in both configurations, syn or anti, with regard to the hydroxyl group on C4 (
      • Nyvall Collén P.
      • Sassi J.-F.
      • Rogniaux H.
      • Marfaing H.
      • Helbert W.
      Ulvan lyases isolated from the Flavobacteria Persicivirga ulvanivorans are the first members of a new polysaccharide lyase family.
      ).
      Protein sequences homologous to LOR_107 were found in Alteromonas sp. LOR (LOR_61) and in two recently sequenced genomes: Alteromonas sp. LTR and Pseudoalteromonas sp. PLSV. Each of these proteins was shown to be an endo-ulvan lyase, producing end products similar to LOR_107. Taken together, these proteins represent the first members of a new family of biochemically characterized polysaccharide lyases. A bioinformatic search using the newly identified protein sequence indicated that many other putative proteins could be members of this novel lyase family. Further, based on the BLASTp results, it seems that this new family is much more abundant in bacterial genomes than the first ulvan lyase family identified in N. ulvanivorans (128 as compared with 20 sequences, respectively, E-value < 1 × 10−4). A key difference between the two ulvan lyase families is the distinctive recognition modalities allowing the LOR ulvan lyase to cleave specifically the glycoside bond between GlcUA and R3S, as opposed to the N. ulvanivorans ulvan lyase that cleaves both GlcUA and IdoUA attached to R3S. Furthermore, the ulvan lyase end products are degraded by β-glucuronyl hydrolase belonging to the GH105 family in N. ulvanivorans (
      • Collén P.N.
      • Jeudy A.
      • Sassi J.-F.
      • Groisillier A.
      • Czjzek M.
      • Coutinho P.M.
      • Helbert W.
      A novel unsaturated β-glucuronyl hydrolase involved in ulvan degradation unveils the versatility of stereochemistry requirements in family GH105.
      ). In contrast, β-glucuronyl hydrolase belonging to the GH88 family appears proximal to the long ulvan lyase of Alteromonas LOR, LTR and Pseudoalteromonas PLSV. This proximity suggests that different enzymes are involved in the degradation of oligo-ulvans. Taken together, these observations raise the possibility that at least two different pathways of ulvan degradation have evolved independently to perform saccharification of ulvan.
      In Alteromonas LOR, Alteromonas LTR, and Pseudoalteromonas PLSV, we found two homologous ulvan lyases: one “short” (59 kDa) and one “long” (110 kDa). The “short” ulvan lyase comprised the catalytic module preceded by a signal peptide, suggesting that it is exported outside the bacterial cell. Interestingly, we were able to show that E. coli can identify the Alteromonas signal peptide and cleave it while secreting a mature and active enzyme to the milieu. The “long” ulvan lyase in addition to the ulvan lyase catalytic module (i.e. the “short” segment) had a conserved type II dockerin repeat domain of about 6.7 kDa at the C-terminal of the protein. Dockerin domains are involved in anchorage of proteins at the cell surface by binding to cohesin (
      • Jindou S.
      • Kajino T.
      • Inagaki M.
      • Karita S.
      • Beguin P.
      • Kimura T.
      • Sakka K.
      • Ohmiya K.
      Interaction between a type-II dockerin domain and a type-II cohesin domain from Clostridium thermocellum cellulosome.
      ). A module of about 48 kDa of unknown function was inserted between the catalytic and dockerin module. In this regard, it is notable that two ulvan lyases were also documented in N. ulvanivorans. The short version is composed of a catalytic module with signal peptide, whereas the long ulvan lyase also comprises a Por secretion system (recently renamed type IX secretion system) C-terminal sorting domain sequence. The C-terminal sorting domain mediates protein secretion by the type IX secretion system, a system that is unique to the Bacteroidetes phylum (
      • Shrivastava A.
      • Johnston J.J.
      • van Baaren J.M.
      • McBride M.J.
      Flavobacterium johnsoniae GldK, GldL, GldM, and SprA are required for secretion of the cell surface gliding motility adhesins SprB and RemA.
      • Sato K.
      • Yukitake H.
      • Narita Y.
      • Shoji M.
      • Naito M.
      • Nakayama K.
      Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.
      ,
      • McBride M.J.
      • Zhu Y.
      Gliding motility and Por secretion system genes are widespread among members of the phylum Bacteroidetes.
      • Shoji M.
      • Sato K.
      • Yukitake H.
      • Kondo Y.
      • Narita Y.
      • Kadowaki T.
      • Naito M.
      • Nakayama K.
      Por secretion system-dependent secretion and glycosylation of Porphyromonas gingivalis hemin-binding protein 35.
      ). Flavobacterium johnsoniae, which also belongs to the Bacteroidetes phylum, utilizes the type IX secretion system both for gliding motility and for extracellular chitinase secretion (
      • Sato K.
      • Naito M.
      • Yukitake H.
      • Hirakawa H.
      • Shoji M.
      • McBride M.J.
      • Rhodes R.G.
      • Nakayama K.
      A protein secretion system linked to bacteroidete gliding motility and pathogenesis.
      ). As genes corresponding to the type IX secretion system apparatus were also found in the N. ulvanivorans genome, the C-terminal sorting domain sequence in the “long” N. ulvanivorans ulvan lyase likely serves to ensure secretion to the medium.
      Full enzymatic conversion of ulvan to monosaccharides requires several more enzymes in addition to ulvan lyase and β-glucuronyl hydrolase, e.g. sulfatase, rhamnosidase, and xylosidase. Employment of a similar polysaccharide utilization locus screening strategy to that reported here may enable discovery of these enzymes. Further mining of the putative proteins may lead to the discovery of novel enzymatic activities and new GH families.

      Author Contributions

      M. K., W. H., A. H., and E. B. designed and analyzed data. M. K, Y. B., and V. B. performed experiments. M. K., W. H., and E. B. wrote the paper.

      Acknowledgments

      We thank Claire Boisset and Laurine Buon from CERMAV for excellent chromatography services and Michael Abeles for support.

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