ι-Carrageenases Constitute a Novel Family of Glycoside Hydrolases, Unrelated to That of κ-Carrageenases*

ι-Carrageenases are polysaccharide hydrolases that cleave the β-1,4 linkages between thed-galactose-4-sulfate and 3,6-anhydro-d-galactose-2-sulfate residues in the red algal galactans known as ι-carrageenans. We report here on the purification of ι-carrageenase activity from the marine bacterium Zobellia galactanovorans and on the characterization of ι-carrageenase structural genes. Genomic libraries from this latter bacterium as well as from Alteromonas fortis were functionally screened for the presence of ι-carrageenase+ clones. The Z. galactanovorans and A. fortis ι-carrageenase genes encode homologous proteins of 53.4 and 54.8 kDa, respectively. Based on hydrophobic cluster analysis and on the 1H NMR monitoring of the products of the overexpressed A. fortisι-carrageenase, these enzymes appear to form a new family of glycoside hydrolases, unrelated to that of κ-carrageenases and with an inverting mechanism of hydrolysis. They both feature a 45-amino acid-long N-terminal segment with sequence similarity to the N-terminal region of several other polysaccharidases. In those for which a three-dimensional structure is available, this conspicuous segment, also deemed “glycanase motif” (Chua, J. E. H., Manning, P. A., and Morona, R. (1999) Microbiology(Reading) 145, 1649–1659), corresponds to a strand-helix-strand “cap” that covers the N-terminal end of a common, right-handed β-helical fold.

The red algal polysaccharides agars and carrageenans exhibit unique rheological properties and are widely used as texturing and moisturizing agents in various industries (1). They consist of a linear backbone of galactopyranose residues linked by alternating ␣(133) and ␤(134) linkages. Although all ␤-linked residues are in the D configuration, the ␣ (1, 4)linked galactose units are in the L configuration in agars and in the D configuration in carrageenans (2). A further layer of complexity is introduced by the occurrence of a 3,6-anhydro bridge in the ␣ (1, 4)-linked galactose residues and by a variable number (from 0 in agarose to 3 in -carrageenan) of sulfate substituents per digalactose repeating unit (Fig. 1). Agars and carrageenans are self-associating polysaccharides, and in the solid or semi-solid state, they readily adopt simple or doublehelice conformations, leading to the formation of aggregates of parallel strands (3).
We maintain various marine bacteria that secrete galactan endo-hydrolases, including producers of -carrageenases, -carrageenases, and -carrageenases as well as of various agarases. These bacteria all belong to the genera Pseudoalteromonas or Alteromonas and to the genus Zobellia, two groups of phylogenetically distant bacteria. All but one of these hydrolases are endo-␤-galactanases (4). They cleave the internal ␤(134) linkages of agars or of carrageenans, yielding oligogalactans of the neocarrabiose or neoagarobiose series (5,6). As these galactan hydrolases display a strict substrate specificity, they obviously recognize the pattern of sulfation on the digalactose repeating units. This set of glycoside hydrolases therefore provides a unique opportunity to investigate structure-function relationships of the hydrolases that degrade sulfated polysaccharides. With this aim, we have undertaken the molecular analysis of a representative set of carrageenases and agarases. The structural genes of the -carrageenase of Pseudoalteromonas carrageenovora and that of another marine bacterium, Zobellia galactanovorans, 1 have been described in detail (7,8). The corresponding -carrageenases were shown to belong to family 16 of glycoside hydrolases as well as the ␤-agarases from Streptomyces coelicolor (8,9) and from Pseudoalteromonas atlantica (GenBank TM accession number M73783) 2 (8). Glycoside hydrolase family 16 (10 -13) also comprises ␤-1,3-glucanases (laminarinases), ␤-1,3-1,4-glucanases known as lichenases, and xyloglucan endotransglycosylases, which catalyze the transfer of segments of ␤-1,4-xyloglucan molecules to other hemicellulose chains in plant cell walls (8,14). Even though -carrageenan differs from agarose by the D configuration of the ␣-linked galactose residues and by the presence of one sulfate substituent at C4 on the ␤-linked D-galactose residues (Fig. 1), -carrageenases and several ␤-agarases belong to the same structural family, and we proposed that these enzymes derived from a common ancestor with laminarinase activity (8). As in other members of this structural family (15), the -carrageenase from P. carrageenovora was shown to proceed with an overall retention of the anomeric configuration and to exhibit transglycosylating activity (16). This enzyme was overproduced and crys-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AJ272071 and AJ272076.
-Carrageenan differs from -carrageenan only by the presence of one additional sulfate substituent per repeating disaccharide, at C2 on the ␣-linked galactose residues (Fig. 1), and one might expect -carrageenases to be structurally related to -carrageenases. However, despite the potential usefulness of -carrageenases in the carrageenan industry and for analyzing the fine structure of carrageenans, this enzyme activity has been purified and investigated in some details only in Alteromonas fortis strain 1 (18), and no -carrageenase gene has yet been characterized. We previously reported that Z. galactanovorans also displays -carrageenase activity (19). Here, we describe the purification and biochemical characterization of this activity as well as the functional cloning of -carrageenaseencoding genes in both A. fortis strain 1 and Z. galactanovorans strain Dsij. Both enzymes cleave the ␤(134) linkages of -carrageenan and display significant sequence similarity to each other. However, they appear unrelated to the ␤-agarases and -carrageenases described above. Moreover, 1 H NMR monitoring of the hydrolysis of -carrageenan by recombinant A. fortis -carrageenase indicates that, unlike ␤-agarases and -carrageenases, -carrageenases are inverting hydrolases. Altogether, they appear as the first representatives of a novel family of glycoside hydrolases.
Purification and Characterization of Z. galactanovorans -Carrageenase--Z. galactanovorans cultures (5 l) were grown, and extracellular proteins were concentrated by tangential ultrafiltration (19). -Carrageenase activity was assayed as follows. Aliquots (100 l) were incubated for 15 min at 40°C with 2 ml of substrate solution consisting of 0.125% (w/v) -carrageenan, 50 mM Tris-HCl, pH 7.2, 5 mM CaCl 2 , 100 mM NaCl, and the reaction mixture (200 l) was assayed for reducing sugars (22) using boiled enzyme as blanks. One unit of enzyme activity (UA) 3 is defined as the amount of enzyme that produces an increase of 0.1 A 237 nm/min in the reducing sugars assay.
Concentrated, cell-free culture supernatants were brought to 30% (w/v) ammonium sulfate and stirred for 1 h. After centrifugation (15,000 ϫ g, 10 min), the supernatant was applied to a column of phenyl-Sepharose CL4B (17 ϫ 1.6 cm) previously equilibrated in 25 mM sodium phosphate buffer, pH 7.5, containing 100 mM NaCl and 30% (w/v) (NH 4 ) 2 SO 4 . The column was washed with buffer to remove the unbound material and eluted with a linear decreasing gradient of (NH 4 ) 2 SO 4 (30 to 0%, 200 ml) at the flow rate of 0.7 ml/min. Eluates were monitored for absorbance at 280 nm, and fractions (4 ml) were assayed for activity towardand -carrageenan. The fractions containing -carrageenase activity were pooled and concentrated with a Centriprep-10 concentrator and analyzed by SDS-PAGE (23) using a 5-12.5% discontinuous polyacrylamide gel. Internal peptide sequences were obtained by Edman degradation (Service de Microséquençage, Institut Pasteur, Paris) from the tryptic digest of acrylamide slices containing about 20 g of purified -carrageenase stained with 0.003% Amido Black. The hydrophobic interaction chromatography fractions retained full enzyme activity for at least 12 months when stored at Ϫ20°C in 5% (NH 4 ) 2 SO 4 and 100 mM NaCl.
Isolation and Analysis of -Carrageenase Clones-Genomic libraries from Z. galactanovorans and A. fortis and the isolation of -carrageenase positive clones were carried out as described previously (7). The genomic libraries each contained approximately 6000 clones. Within 1 week of culture at 22°C on Zd -carrageenan broth, five independent colonies from the Z. galactanovorans library, referred to as pIC1-5, as well as two clones from that of A. fortis (pIP1-2) made a hole in the substratum. The inserts from the colonies harboring -carrageenase activity were submitted to single and double digestion with various restriction enzymes and mapped by agarose gel electrophoresis. The pIP1 and pIP2 plasmids from A. fortis library harbored inserts of 10.4 and 4.1 kb, respectively, which shared a common HindIII-XbaI fragment of 3.0 kb. The inserts from Z. galactanovorans pIC1-5 plasmids ranged in size from 8.0 to 16.4 kb, and they all shared a common PvuII fragment of 3.3 kb. This fragment was sub-cloned into pBluescript KSII phagemid (Stratagene) in both orientations (pICP07 and pICP16). To further localize the -carrageenase gene, an ExoIII library was built from pICP07 plasmid using the double-stranded nested deletion kit (Amersham Pharmacia Biotech). Two ExoIII clones, referred to as pICP07.4 (1400-bp insert) and pICP07.12 (1800-bp insert), displayed a positive phenotype on Zd broth solidified with -carrageenan.
Sequencing was carried out using the dideoxy-sequencing method (24) with T7 DNA (Amersham Pharmacia Biotech) with synthetic oligonucleotide as primers. Hydropathy was analyzed with the Translate program of MacMolly package using the Kyte and Doolittle algorithm (25). -Carrageenase amino acid sequences were aligned using the Gap

FIG. 1. Structures of red algal-sulfated galactans.
A, disaccharide repeating units of agarose and of various carrageenans. Note that agarose is a neutral galactan, whereas -, -, and -carrageenans are substituted by one, two, and three sulfate groups per repeating disaccharide unit, respectively. B, -neocarrahexaose sulfate, i.e. the hexasaccharide made of three unit repeats of -carrageenan (DP3). In the nomenclature designed by Knutsen and Grasdalen (31,32) to describe the fine structure of carrageenans, A and G refer to 3,6-anhydrogalactose and to galactose residues, respectively, and S refers to sulfate substituents, whereas nR and R indicate the non-reducing and reducing ends, respectively. Accordingly, the disaccharide at the non-reducing end comprises 3,6-anhydro-␣-D-galactopyranose 2-sulfate and ␤-D-galactopyranose 4-sulfate, which are designated as A2S nR and G4S nR; the internal 3,6-anhydro-␣-D-galactopyranose 2-sulfate and ␤-D-galactopyranose 4-sulfate residues are referred to as A2S and G4S; at the reducing end, 3,6-anhydro-␣-D-galactopyranose and ␣or ␤-D-galactopyranose 4-sulfate are indicated by A2S R and G4S R ␣/␤. program (Wisconsin Package, version 8, Genetics Computer Group, Madison, WI), with a gap creation penalty of 3.0 and a gap extension penalty of 0.1. Hydrophobic cluster analysis (26) was performed with the help of the DRAWHCA program (27). The three-dimensional structure was rendered using the programs MOLSCRIPT (28) and RASTER3D (29).
Production of Recombinant -Carrageenase-The procedure and results of the overexpression of the -carrageenase gene from A. fortis are described in detail elsewhere (39). Briefly, an in-phase insertion into the expression vector pET20b of the coding region of A. fortis -carrageenase followed by a polyhistidine tail was constructed by polymerase chain reaction. The polymerase chain reaction-generated products were sequenced to check the construct accuracy, and cultures conditions were then developed for overexpression in the presence of isopropyl-1-thio-␤-D-galactopyranoside, resulting in yields of -carrageenase activity of ϳ75 units ml Ϫ1 in the culture medium. Nickel column-purified fractions of the recombinant -carrageenase were used to monitor the hydrolysis kinetics of -carrageenan (see below).
Analysis of -Carrageenase Products-Enzymic hydrolysis of -carrageenan was performed as described by Rochas and Heyraud (5) using 0.2 UA/mg of polymer, and the end-products were analyzed by 13 C NMR (6). Briefly, the enzyme-resistant fraction was precipitated with 70% (v/v) isopropanol, and the soluble oligosaccharide fraction was fractionated on Bio-Gel P6 in an Amicon column (95 ϫ 4.4 cm, 25°C). The eluant was 0.03 M NaNO 3 , and detection was performed with a Helma ERC 5710 differential refractive index monitor. Hydrolysis kinetics were monitored by carbohydrate polyacrylamide gel electrophoresis (30) using 6% (w/v) polyacrylamide for the stacking gel, 18% polyacrylamide for the running gel, and 2% piperazine diacrylamide as reticulating agent. Gels were stained in a 0.5% Alcian Blue solution for 30 min, then with silver nitrate. Bio-Gel P6-purified -carrageenan oligomers with a known degree of polymerization (DP, expressed as the number of disaccharide repeating units) were used as size markers.
1 H NMR Spectroscopy Analysis of Hydrolysis Mechanism-Desalted -carrageenan (12 mg) was dissolved in 1 ml of 99.95% D 2 O and freezedried. The procedure was repeated three times to replace all the exchangeable protons by deuterium. The resulting polysaccharide was placed in a dry, 5-mm NMR tube. 1 H spectra were recorded at 25°C with a Varian Unity Plus 500 spectrometer operating at 500 MHz, equipped with an ␣ЈH-X PFG inverse probe and a temperature-controlling unit. Each experiment was run with a sweep width of 1750 Hz and a time domain of 12,416 words, resulting in an acquisition time of 3.547 s. The pulse width was 10.3 ms, and the number of scans was 32. Each free induction decay was zero-filled to 32,768 words before Fourier transformation. A presaturation was done for 1.5 s before the acquisition at the frequency of H 2 O (Ϫ159.8 Hz), with a power of Ϫ9 dB. After recording the spectrum of the substrate, 100 l of recombinant -carrageenase from A. fortis (6 mg/ml in 99.95% D 2 O) were added to the NMR tube, which was immediately placed back in the spectrometer. Proton NMR spectra were recorded every 210 s for 3 h then at 24 h after the addition of the enzyme, i.e. when enzyme hydrolysis was complete and the mutarotation equilibrium was reached. NMR signals were assigned according to Knutsen and Grasdalen (31,32) and based on a COSY (correlation spectroscopy) spectrum of oligo -carrageenans (data not shown).

RESULTS
Purification and Characterization of the -Carrageenase from Z. galactanovorans-The fractionation of Z. galactanovorans culture supernatants by hydrophobic interaction chromatography on phenyl-Sepharose CL4B resolved the -carrageenase and -carrageenase activities, typically resulting in a 8-fold partial purification of -carrageenase with a 16% recovery (Table I). Based on SDS-PAGE analysis ( Fig. 2A) and assuming that activities are proportional to the protein band intensities, the -carrageenase was likely to correspond to the band with an apparent molecular mass of ϳ50 kDa. This assumption was supported by gel permeation chromatography experiments (data not shown), which indicated a molecular mass of about 50 kDa for the native -carrageenase. Accordingly, the SDS-PAGE 50-kDa protein band was excised, and internal peptide microsequences were determined by Edman degradation (underlined in Fig. 3).
Based on the time course of the degradation of -carrageenan by Z. galactanovorans, the enzyme behaved as an endo-hydrolase, rapidly yielding low molecular weight -carrageenan oligomers. As confirmed by 13 C NMR spectroscopy (not shown), the major end products were -neocarratetraose sulfate and -neocarrahexaose sulfate (Fig. 2B). The partially purified -carrageenase fractions were active neither on purifiedand -carrageenans nor on agar and agarose.  The -Carrageenase Gene from Z. galactanovorans-Plasmid pICP07.12 was used to determine on both strands the nucleotide sequence of the structural gene of the -carrageenase from Z. galactanovorans. The insert, 1837 bp in length, contained a single open reading frame of 1473 bp, referred to as cgiA (Fig.  3). In the 5Ј non-coding region, 2 invert-repeated, 26-bp-long sequences are found, possibly corresponding to a hairpin structure with a free energy of Ϫ38.5 kJ/mol. Two hexamers, TgGAag and TATAAT, consistent with E. coli Ϫ10 and Ϫ35 consensus promoters and separated by 17 nucleotides are present 118 nucleotides upstream of the start codon (ATG 333 ). No canonical Shine-Dalgarno sequence can be identified immediately upstream of the initiation codon. However, as already described for the Z. galactanovorans -carrageenase gene (8), sequences referred to as ⑀ and are present both upstream and downstream of ATG codon, which may serve as a ribosome binding site (33). Using the pIC1 plasmid, 160 bp were further sequenced downstream of the stop codon (TGA 1806 ), unraveling a putative stem loop with a predicted free energy of Ϫ109 kJ/mol followed by four thymidine residues that may represent a -independent transcriptional termination site. Interestingly, a second open reading frame, with its start codon (ATG 1866 ) also preceded by and ⑀ sequences, began immediately after this putative termination stem loop.
The predicted product of Z. galactanovorans -carrageenase gene is a protein of 491 amino acids, with a theoretical molecular mass of 53.4 kDa (Fig. 3). It includes the two internal peptides determined by microsequencing of the 50-kDa protein band excised from the SDS gel shown in Fig. 2A. As indicated by hydropathy analysis (not shown), 12 N-terminal amino acids of the protein stand out as a domain with a high hydrophobicity, suggesting that they belong to a signal peptide (34 -36). The CgiA N terminus indeed harbors a signature typical of the N termini of prokaryotic, outer membrane lipoproteins, LASI-AIMAIGCTK, with a putative cleavage site by signal peptidase II between the Gly 19 and Cys 20 residues (Prosite PS00013 (37)).
The computed molecular mass of the resulting mature protein, 51.32 kDa, is similar to the molecular mass of the purified protein, as determined by SDS-PAGE (50 kDa, Fig. 2).
The -Carrageenase Gene from A. fortis-The 3.0-kb HindIII-XbaI fragment common to pIP1 and pIP2 plasmids from A. fortis library was subcloned and used to determine on both strands the nucleotide sequence of the structural gene of A. fortis -carrageenase (Fig. 4). It contained a single open reading frame of 1473 bp. Two hexamers, TTGctt and TATAAa, 41 nt upstream of the start codon (ATG 211 ) and separated by 20 nucleotides, may correspond to Ϫ35 and Ϫ10 boxes. A typical Shine-Dalgarno sequence (GGAG) is indeed present 7 nt upstream of the initiation codon. Downstream of the stop codon (TAG 1684 ), a hairpin structure with a free energy of Ϫ93.6 kJ/mol and followed by 6 T residues, may function as a site for -independent transcriptional termination. The A. fortis cgiA gene is followed by a second, partial open reading frame (ATG 1880 ), itself endowed with putative TTGttA and TATAtT Ϫ35 and Ϫ10 promoter sequences and with a typical Shine-Dalgarno sequence (GGAG).
The predicted product of the -carrageenase gene from A. fortis is a protein of 491 amino acids, with a theoretical molecular mass of 54.80 kDa (Fig. 4). The N terminus of the protein displays a high hydrophobicity (not shown), characteristic of a signal peptide, with a putative cleavage site between Gly 26 and Ala 27 (38). Accordingly, the molecular mass of the mature protein is 51.95 kDa, a value similar to the value determined by SDS-PAGE analysis, 57 kDa (18). A polymerase chain reaction product corresponding to the mature protein was overexpressed in E. coli, using pET20b as expression vector. Yields typically ranged from 60 to 75 UA/ml, allowing for the purification and crystallization of the recombinant -carrageenase (39).
The -Carrageenase from A. fortis Proceeds with an Overall Inversion of the Anomeric Configuration-The hydrolysis of -carrageenan by the overproduced recombinant A. fortis -car- rageenase was monitored using 1 H NMR spectroscopy. Compared with the 1 H NMR spectrum of -carrageenan, the spectra recorded from the reaction mixture, including as early as 7 min after the addition of the enzyme, showed several new resonances (Fig. 5). Several of those signals can be assigned to newly formed reducing ends, i.e. those from 3,6-anhydro-␣-Dgalactopyranose-2-sulfate (A2S R ␣) and ␣-D-galactopyranose-4-sulfate (G4S R ␣) residues. They include, for example, the signals that we assign to G4S H4 R ␣ (4.84 ppm), G4S H1 R ␣ (5.20 ppm), and G4S H2 R ␣ (3.84 ppm), which appeared and increased with the onset of hydrolysis. By contrast, the intensity of the signal of G4S H2, whose chemical shift (3.50 ppm) is almost identical to G4S H2 R ␤, decreased over the same period. The spectra recorded from 15 min to 24 h after the addition of the enzyme showed significant changes relative to the earlier ones. The signals attributable to ␤-anomers (for instance G4S H4 R ␤, G4S H1 R ␤, G4S H3 R ␤, and A2S H1 R ␤) became predominant, whereas those of the ␣-anomers (for instance G4S H4 R ␣, G4S H2 R ␣, and A2S H1R ␣) decreased.

DISCUSSION
The -Carrageenases from Z. galactanovorans and A. fortis Are Homologous-The -carrageenases from Z. galactanovorans (Fig. 2B) and from A. fortis (18) each release -neocarratetraose sulfate (DP2) and -neocarrahexaose sulfate (DP3) as their main end products. Hence, these two hydrolases cleave the ␤(134) linkages of -carrageenan, and -neocarraoctaose sulfate (DP4) is the shortest substrate oligomer that can be cleaved. Both Z. galactanovorans -carrageenase (Fig. 2B) and that from A. fortis (data not shown) tend to preferentially release small -carrageenan fragments, suggesting a processive behavior. As indicated by the solubilization properties of A. fortis -carrageenase (18) and by the signal peptide configuration of the -carrageenase of Z. galactanovorans, both enzymes are likely localized in the bacteria outer membranes.
Since E. coli is devoid of -carrageenase activity, our strategy to clone these enzymes was based on the ability of E. coli colonies recombined with Z. galactanovorans or A. fortis genomic DNA to degrade -carrageenan. Several lines of evidence indicate that we did clone the Z. galactanovorans and A. fortis -carrageenases by this functional cloning approach: (i) the molecular masses predicted by the CgiA amino acid sequences are consistent with those determined by SDS-PAGE analysis of the -carrageenases purified from the culture supernatants; (ii) two internal peptides characterized by microsequencing of Z. galactanovorans -carrageenase protein band ( Fig. 2A) are present in the relevant CgiA gene (Fig. 3); and (iii) the overexpressed A. fortis CgiA protein exhibited a specific -carrageenase activity of 5000 UA/mg (39).
As shown by Fig. 6, both enzymes are very similar in their primary structures, with 43 and 59% identity and similarity, respectively. A. fortis is a ␥-proteobacterium, whereas Z. galactanovorans belongs in the family Flavobacteriaceae (order Cytophagales), two phylogenetically distant groups of bacteria, yet their -carrageenases are markedly related, both in their structure and in their catalytic properties, suggesting that they have arisen by speciation from a common ancestor. The three best conserved domains are in the central region, from Lys 182 to Val 200 , Gly 227 to Leu 259 , and Val 286 to Met 293 (numbering of the Z. galactanovorans sequence). Enzymatic hydrolysis of glycosidic bonds is known to occur via two general mechanisms leading either to retention or to inversion of the anomeric configuration. Both mechanisms require two critical amino acids, most often glutamic or aspartic acid residues (40 -43). Nine aspartic acid residues and five glutamic acid residues are strictly conserved in the two proteins, including three of each localized in the conserved domains. Such invariant Asp and Glu residues are good candidates for acting as catalytic residues. -Carrageenases Form a Novel Family of Glycoside Hydrolases-Consistent with the sequence similarities pointed out in Fig. 6, the hydrophobic cluster analysis plots of Z. galactanovorans and A. fortis -carrageenases (not shown) exhibited a similar distribution of the hydrophobic clusters along the proteins. The hydrophobic cluster analysis score based on these correspondences was 82% over 293 amino acids, well above the similarity threshold in this comparison procedure (44). Secondary structure predictions using JPRED (45) on the two -carrageenases suggest that these enzymes are mostly composed of ␤-strands with few ␣-helices (not shown). With the exception of a ϳ45-amino acid-long N-terminal segment (see below), no significant similarity was found between -carrageenases and other known proteins. Taken together, these results indicate that Z. galactanovorans and A. fortis -carrageenases are, so far, the only two members of novel family of glycoside hydrolases. The classification of glycoside hydrolases (10 -13), glycosyltransferases (46), and polysaccharide lyases 4 is available on the World Wide Web.
Interestingly, the consensus, so-called glycanase motif (47) DFGX 3 DGX 6 AX 3 A, shared by various prokaryotic and eukaryotic polysaccharide-degrading or -modifying enzymes such as bacteriophage K1E tailspike protein, bacteriophage Sf6, endorhamnosidase, Azotobacter vinelandii mannuronan-C5-epimerase, and several family 28 polygalacturonases, is also present in A. fortis -carrageenase and, although more divergent, in the Z. galactanovorans CgiA sequence. A closer inspection of the sequences of these proteins using the hydrophobic cluster analysis method (26,27) shows that the "glycanase motif" can be extended over ϳ45 amino acids (from Val-52 to Gly-96 in A. fortis -carrageenase) (data not shown). However, the threedimensional structure of three family 28 glycoside hydrolases, namely rhamnogalacturonase A from Aspergillus aculeatus (48) and polygalacturonases from Erwinia carotovora and Aspergillus niger (49,50), have been determined, and the region corresponding to the glycanase motif above forms a strandhelix-strand "cap," which covers the N-terminal end of the common right-handed ␤-helical fold shared by the three proteins. This cap is located far away from the active site cleft of the enzymes, and it is difficult to imagine that it may fulfil a substrate binding function.
The -Carrageenases Are Structurally and Catalytically Unrelated to -Carrageenases--Carrageenan differs from -carrageenan only by the presence of an additional sulfate group on the digalactose repeating unit, at the C2 position of the ␣ (1,4)-linked galactose residues (Fig. 1A). Yet we show here that the -carrageenases are totally unrelated to -carrageenases in their amino acid sequences and, by consequence, most likely so at the three-dimensional level. In particular, the -carrageenases lack the motif E[ILV]/[IVAF][VILMF](0.1)E, i.e. the catalytic site typical of clan B of glycoside hydrolases (51)(52)(53), which encompasses glycoside hydrolase families 7 and 16 (13). Moreover, 1 H NMR analysis of the anomeric configuration of the hydrolysis products of the -carrageenase from A. fortis indicates that this enzyme inverts the anomeric bond configuration, producing ␣-anomers that progressively give rise to ␤-anomers when mutarotation takes place (Fig. 5). Stereochemistry of hydrolysis is dictated by the fine topology of the 4 B. Henrissat, unpublished data.
FIG. 5. 1 H NMR monitoring of the hydrolysis of -carrageenan by A. fortis recombinant -carrageenase. Deuterated -carrageenan (12 mg) was incubated at 25°C in the presence of 300 UA of recombinant -carrageenase. At intervals, 1 H NMR spectra were recorded for 210 s. Peak assignments are labeled according to the nomenclature of Fig. 1B  (see the inset). Note that the 1 H resonances of A2S residues, such as A2S R ␤, were affected by the ␣/␤ equilibrium on the neighboring G4S ␣/␤-reducing ends.
FIG. 6. Sequence alignment of Z. galactanovorans (lower sequence) and A. fortis (upper sequence) -carrageenases. Arrows and stars indicate the invariant aspartic acid and glutamic acid residues, respectively. The signature for the occurrence of a right-handed parallel ␤-helix fold is boxed. active sites, and the hydrolytic mechanism is known to be absolutely conserved within glycoside hydrolase families (13,54). It follows that Z. galactanovorans and A. fortis -carrageenases both hydrolyze the ␤(134) linkages of -carrageenan by a one-step nucleophilic substitution, a mechanism that results in the inversion of the anomeric configuration and precludes transglycosylation (41). This finding contrasts with the case of -carrageenases, which are retaining glycoside hydrolases with transglycosylating properties (16).
Altogether, we show here thatand -carrageenases exhibit different active sites, hydolytic mechanisms, and presumably overall folds. That it takes completely different proteins to hydrolyzeand -carrageenans suggests that the sulfate substituents at the C2 position of the ␣ (1, 4)-linked galactose residues of these galactans are major determinants for substrate specificity and/or hydrolysis. This results substantiates our working hypothesis that, in the evolution of agarases and carrageenases, different structural features have been selected to accommodate the linear density and position of sulfate groups on galactans. At this stage, however, we do not rule out the possibility that, beyond their chemical structure, the threedimensional structures of these sulfated galactans in the solid state (helices or agregates of double helices) also are critical in the interactions with the enzymes catalyzing their hydrolysis.