Alpha-Agarases Define a New Family of Glycoside Hydrolases, Distinct from Beta-Agarase Families

The gene encoding the ${alpha}$ -agarase from "Alteromonas agarilytica"(proposed name) has been cloned and sequenced. The gene product(154 kDa) is unrelated to ß-agarases and instead belongsto a new family of glycoside hydrolases (GH96). The ${alpha}$ -agarasealso displays a complex modularity, with the presence of fivethrombospondin type 3 repeats and three carbohydrate-bindingmodules.

INTRODUCTION

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INTRODUCTION

Agars are the main cell wall components of numerous red macroalgae.These polymers consist of 3,6-anhydro-L-galactoses and D-galactosesalternately linked by ${alpha}$ -(1,3) and ß-(1,4) linkages(10). Agars constitute a crucial carbon source for a numberof marine bacteria which secrete agarolytic enzymes, mainlyß-agarases (EC 3.2.1.81), which hydrolyze the ß-(1,4)linkages (for a review, see reference 12). They are found inthree distinct families of glycoside hydrolases (GH), familiesGH16, GH50, and GH86 (CAZY database [6]). Structural data areavailable only for the GH16 ß-agarases from Zobelliagalactanivorans, AgaA_Zg and AgaB_Zg (1, 2, 9). In contrast, "Alteromonasagarilytica" (proposed name) secretes an ${alpha}$ - agarase (EC 3.2.1.158)which randomly hydrolyzes the ${alpha}$ -(1,3) linkages in agars, releasingagarotetraose as its main end product (13, 15, 18). In thiscontext, we sought to determine whether the agarases of the ${alpha}$ type share any structural relationships with ß-agarases.We report here the cloning of the ${alpha}$ -agarase gene agaA from "A.agarilytica," and we demonstrate that ${alpha}$ -agarases define a newGH family.

Cloning of the agaA gene.

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Cloning of the agaA...

The ${alpha}$ -agarase from "A. agarilytica" (AgaA_Aa) was purified aspreviously described (13), and N-terminal and internal peptidesequences were determined (Pasteur Institute, France) as follows:(i) ETLELQAESFANSGGFF and (ii) QPRVYNPNEHIVAEIQGPAT, respectively.With degenerated oligonucleotides derived from these microsequences(CARGCIGARTCITTYGCIAA and TAYAAYCCNAAYGARCAYAT, respectively),a 2.5-kb DNA fragment was amplified by PCR using "A. agarilytica"genomic DNA and labeled with [ ${alpha}$ -³²P]dCTP. This radiolabeled probewas used to screen an "A. agarilytica" genomic library, preparedas previously described (5). Among the $~$ 5,000 recombinant clones,two positive clones (pAA1 and pAA2) were identified, with insertsof 7.4 kb and 17.9 kb in length, respectively. Southern blottinganalysis and plasmid mapping indicated that both inserts encompassedthe same gene, which is present in only one copy in the genome.Plasmid pAA1 was sequenced on both strands over 4,651 bp, anda single open reading frame was identified and referred to asagaA (4,287 bp). Potential –35 and –10 promoterregions (TTGATC and TACACA, respectively) and a Shine-Dalgarnosequence (GGAG) were identified upstream of the start codon.A possible transcription termination codon was found downstreamof the TAA stop codon. The deduced gene product is a preproteinof 1,429 residues (154 kDa) which includes the peptides A andB. A signal peptide cleaved between A26 and E27 is predictedby SIGNALP (4), consistent with the N-terminal sequencing ofthe purified extracellular ${alpha}$ -agarase.

Several attempts were made to overexpress AgaA_Aa and its isolatedmodules with Escherichia coli, with pET or pGEX vectors, undervarious conditions. Unfortunately, the constructs always yieldedinclusion bodies. However, within 1 week of culture at 22°Con Zd agar broth (3), the E. coli clones harboring the plasmidpAA1 dug a hole in the substratum, indicating agar degradation.Therefore, under the control of its own promoter, agaA was successfullytranslated into an active, recombinant enzyme, confirming thatthis gene indeed encodes the ${alpha}$ -agarase.

AgaA_Aa is a complex, modular protein with a catalytic domain that defines a new GH family.

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AgaA_Aa is a complex,...

Only the N-terminal region of AgaA_Aa displays significant sequencesimilarity with proteins in the UniProt database. Based on InterProScan(14) analysis, eight distinct modules were identified in thisregion (Fig. 1A), including five thrombospondin type 3 (TSP3)repeats (TSP3-1 [amino acids D171 to G203], TSP3-2 [D360 toL392], TSP3-3 [D393 to A425], TSP3-4 [D426 to L458], and TSP3-5[D459 to G491]) and three carbohydrate-binding modules fromfamily 6 (CBM6-1 [amino acids E27 to R159], CBM6-2 [E209 toT343], and CBM6-3 [S659 to L792]).

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FIG. 1. (A) Modular architecture of the ${alpha}$ -agarase AgaA_Aa. CBM6 and GH96 refer to carbohydrate-binding modules of family 6 and the glycoside hydrolase module of family 96, respectively. Gray boxes correspond to the TSP3 repeats. (B) Structure-based alignment of the TSP3 repeats of the human thrombospondin (PDB code 1UX6), of the ${alpha}$ -agarase AgaA_Aa, and of the cellulase CelG from Pseudoalteromonas haloplanktis (trEMBL code O86099). (C) Structure-based alignment of the three CBM6s from the ${alpha}$ -agarase AgaA_Aa and of the agar-specific CBM6s tethered to the ß-agarases Aga16B_Sd (GenPept no., ABD80437) and Aga86E_Sd (GenPept no., ABD81915) from Saccharophagus degradans. These modules are compared to the secondary structures of the CBM6 appended to Aga16B_Sd (PDB code 2CDO). Alpha helices and beta strands are represented as helices and arrows, respectively, and beta turns are marked with TT. The black triangles mark the residues involved in the recognition of the nonreducing end of agarose chains. Figure 1B and C were prepared using ESPrit software (7) and use the same shading codes.

The characterized protein that most closely matches the TSP3repeats of AgaA_Aa is the cellulase CelG from Pseudoalteromonashaloplanktis. In CelG, the TSP3 repeats constitute an extendedlinker connecting the GH5 catalytic module and a C-terminalCBM5 (17). The TSP3 repeats of AgaA_Aa present about 50% sequenceidentity with their counterparts in CelG. They also display $~$ 30% sequence identity with the "true" type 3 repeats found inhuman thrombospondin, whose crystal structure has been solved(11). These modules lack secondary structures and are organizedaround a core of calcium ions coordinated by conserved aspartates[DXDXDGXX(D/N)XXDXC motif]. The conserved cysteine is involvedin a disulfide bridge linking adjacent TSP3 repeats, strengtheningtheir stability (11). This motif is strictly conserved in eachof the TSP3 repeats of AgaA_Aa (Fig. 1B), indicating that thesemodules adopt a similar structure and likely bind calcium ions.This is consistent with the observation that ${alpha}$ -agarase activityis stabilized by the presence of calcium ions (13, 18).

In BLASTp searches with the three CBM6 modules from AgaA_Aa,the highest E values are always obtained for the CBM6 sequencestethered to ß-agarases, while they decrease significantlywith CBM6 linked to nonagarolytic enzymes. Only the CBM6s attachedto the ß-agarases from Saccharophagus degradans, Aga16B_Sdand Aga86E_Sd, were shown to actually bind agarose (8). A pair-wisecomparison indicates a strong sequence identity (51%) betweenthe CBM6-1 and the CBM6 from Aga16B_Sd (Fig. 1C), while thoseof CBM6-2 and CBM6-3 are more divergent (28% and 26%, respectively).The crystal structure of the CBM6 from Aga16B_Sd revealed thatfive residues are critical for the recognition of the nonreducingend of the agarose chain, Asn39, Tyr40, Trp97, Trp127, and Asn130(8). Four of these residues are strictly conserved in CBM6-1,while Tyr40 is replaced by a similar aromatic amino acid, Phe64(Fig. 1C). Together, these results strongly suggest that CBM6-1is an agar-binding module and likely displays selectivity towardthe nonreducing termini of agarose chains. In contrast, thefive critical residues are only partially conserved in CBM6-2and CBM6-3. Therefore, the specificities of these CBM6 sequencesare less certain.

Finally, a BLASTp search of the patent data bank at NCBI identifiedtwo proteins from a marine bacterium with a strong sequenceidentity with the C-terminal region of AgaA_Aa (49% and 77%;Fig. 2). These proteins, also described as ${alpha}$ -agarases (16), encompassthree modules, two N-terminal CBM6s and the C-terminal moduleconserved with AgaA_Aa. Since the N-terminal region of AgaA_Aaencompasses only additional, noncatalytic modules, its conservedC-terminal region (Asn809 to His1429) likely contains its activesite. Together, these three catalytic modules ( $~$ 620 residues)constitute a new family of glycoside hydrolases, referred toas family GH96 (CAZY database). Therefore, ${alpha}$ -agarases are structurallyunrelated to the ß-agarases from the families GH16,GH50, and GH86.

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FIG. 2. Sequence alignment of the catalytic module of AgaA from A. agarilytica and of Aga14 and Aga15 (16). Shaded boxes enclose conserved positions. Figure 2 was prepared using ESPrit software and the same shading codes as seen in Fig. 1B and C.

Accession numbers.

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Accession numbers.

The nucleotide sequence of the ${alpha}$ -agarase gene from "Alteromonasagarilytica" has been deposited in GenBank with the accessionnumber AF121273. Its amino acid sequence is available in Swiss-Protunder the accession number Q9LAP7.

ACKNOWLEDGMENTS

We gratefully acknowledge the help of the Conseil Régionalde Bretagne, which supported this research through a Ph.D. fellowshipto D.F. and a research grant (PRIR number 98C389).

FOOTNOTES

* Corresponding author. Mailing address: Centre National de la Recherche Scientifique, Université Pierre et Marie Curie-Paris6, Unité Mixte de Recherche 7139, Station Biologique, F-29682 Roscoff Cedex, Bretagne, France. Phone: 33 298 29 23 30. Fax: 33 298 29 23 24. E-mail: gurvan{at}sb-roscoff.fr

Published ahead of print on 18 May 2007.

Present address: UMR 6197 (IFREMER/CNRS), Laboratoire de Microbiologieet Environnements Extremes, Plouzane, Bretagne, France.

REFERENCES

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