3.1 Sequence characteristics of the alginate lyases.
ORF2549 in the genome of Flammeovirga sp. strain MY04 was predicted to encode a candidate polysaccharide lyase, Aly-6 (GenBank Protein Accession No. ANQ49918.2). The gene is 2,238 bp in full-length with a GC content of 36.8%. The putative protein Aly-6 is composed of 745 amino acid residues with an apparent molecular mass of 84.67 kDa. The predicted pI value is 7.30. SignalP 5.0 analysis indicated that the signal peptide of Aly-6 contains 23 amino acid residues (Met1 to Ser23) (Fig. 1C).
Analyses using the Carbohydrate-Active Enzyme database and the Simple Modular Architecture Research Tool indicated that the Aly-6 protein contains a putative N-terminal catalytic module (Ala76 to Phe320) associated with PL5 alginate lyases (AlgL), as well as a putative C-terminal module (Leu391 to Val664) associated with heparinase II or III (Hep II_III) from Pedobacter heparinus (Fig. 1C). BLASTp searches showed that among characterized enzymes, the whole Aly-6 protein shares the greatest sequence identity (38.93%) with the exo-type alginate lyase Alg17c from Saccharophagus degradans strain 2–40, followed by 38–35% identities with four other reported PL17 family alginate lyases (Fig. 1D): OalS17 of Shewanella sp. strain Kz7, AlgL of Sphingomonas sp. strain MJ-3, OAL of Stenotrophomonas maltophilia, and AlyII of Pseudomonas sp. strain OS-ALG-9. Subsequently, the full-length Aly-6 protein shares very low sequence identities with PL15 and PL5 family alginate lyases (Table. S2). The PL17 proteins described above are all organized in an AlgL and Hep_II_III complex modular architecture similar to that of Aly-6. Notably, the whole Aly-6 protein shares no homology with studied PL6 or PL7 alginate lyases, including Aly1, Aly2, and Aly5 from the same Flammeovirga strain MY04, which contains neither AlgL-like nor Hep_II_III-like modules.
Furthermore, protein sequence alignment showed that the AlgL module of Aly-6 contains one putative catalytic motif, H195-N196-H197-S198-T199-W200, which is half-conserved but different from the conserved NNHSYW catalytic motif of diverse PL5 alginate lyases and the conserved HNHG(A)TW catalytic motif of various PL17 alginate lyases (Fig. S1). Phylogenetic analysis indicated that Aly-6, together with the six genome-predicted alginate lyases that are organized in the same AlgL and Hep_II_III complex modular architecture, i.e., four proteins of other Flammeovirga strains, one of Roseivirga ehrenbergii, and one of R. echinicomitans, are clustered into a novel separate branch within the PL17 superfamily (Fig. 1D).
3.2 Production and purification of the full-length and truncated recombinant proteins.
The full-length Aly-6 gene was amplified directly from the genomic DNA of Flammeovirga sp. strain MY04. The 2.3-kb PCR products produced by the primers E30Aly-6-F and E30Aly-6-R were gel-recovered and enzyme-cloned into the pET30a (+) vector downstream of a T7 promoter. Thus, a His×6 tag was successfully added to the C-terminus of the protein product (rAly-6) encoded by the resulting expression vector (pET30-Aly-6). Similar operations were performed on the PCR products produced by three primer pairs, TF-Aly-6-F and TF-Aly-6-R, TF-Aly-6-Lm-F and TF-Aly-6-Lm-R, and TF-Aly-6-Hpm-F andTF-Aly-6-Hpm-F, to obtain the expression vectors pCTF-Aly-6, pCTF-Aly-6-Lmodule, and pCTF-Aly-6-HPmodule, which added a His×6 tag followed by a TF factor to the N-terminus of the full-length protein product (rTF-Aly-6) and two truncated protein products (rTF-Aly-6-Lmodule and rTF-Aly-6-Hpmodule) individually, thus generating recombinant fusion proteins. To express and purify the recombinant proteins Pae-rAlgL and Avi-rAlgL, similar operations were performed using the pET30a (+)-derived recombinant vectors pE30-AlgL-Pae and pE30-AlgL-Avi, which added a His×6 tag at the C-terminus of the AlgL-Pae and AlgL-Avi proteins, respectively.
SDS-PAGE analyses indicated that BL21(DE3) cells harboring each of the above recombinant plasmids produced soluble proteins (Fig. S2), with the correct apparent molecular mass and yields greater than 1.0 g/liter. After sonication and centrifugation, crude enzymes were each extracted from the E. coli cultures. The soluble protein fractions containing rAly-6, rTF-Aly-6, rTF-Aly-6-Lmodule, rTF-Aly-6-Hpmodule, Pae-rAlgL, and Avi-rAlgL were eluted from a Ni-nitrilotriacetic acid (NTA) column using imidazole at concentrations above 50 mM. Further SDS-PAGE analyses indicated that the purified soluble proteins each had purities greater than 99% and initial concentrations greater than 1.0 mg/ml (Fig. S2).
3.3 Enzyme characteristics of the recombinant proteins.
The recombinant proteins rAly-6 and rTF-Aly-6 showed the same substrate spectrum, i.e., they did not digest chondroitin, chondroitin sulfates (A, C, D, and E types), dermatan sulfate B, hyaluronan, heparin, heparin sulfate, pectin, or xanthan, but they efficiently digested alginate, M blocks, and G blocks to produce oligosaccharide products exhibiting strong absorbance at 235 nm. The results suggested that the Aly-6 protein of Flammeovirga sp. strain MY04 is a bifunctional alginate lyase. Furthermore, enzyme activity tests of rAly-6 and rTF-Aly-6 indicated a similar substrate preference for M over G (Table. 1). Quite different from the whole proteins rAly-6 or rTF-Aly-6, the recombinant soluble truncated Aly-6 proteins, i.e., rTF-Aly-6-Lmodule and rTF-Aly-6-HPmodule, showed little degradation activity against any tested polysaccharides, including alginate-associated substrates (Table. 1), indicating that both the AlgL-like and the Hep_II_III-like modules are integral for the alginate lyase activity of the whole Aly-6 enzyme of Flammeovirga sp. strain MY04. Furthermore, the above results demonstrated that in the Aly-6 protein, the Hep_II_III-like module is only a putative element instead of a catalytic module against any tested glycoaminoglycans, including heparin or heparin sulfates. The purified protein fractions of Pae-rAlgL and Avi-rAlgL could efficiently degrade only alginate and M blocks to generate unsaturated oligosaccharide products, and they could hardly digest any other tested polysaccharides, including G blocks (Table. 1), demonstrating that they are M-specific lyases, as reported previously. In addition, none of the above protein preparations degraded acylated alginate from Azotobacter or Pseudomonas bacterial strains to produce detectable oligosaccharide products.
The whole enzyme rAly-6 demonstrated the highest activity at 40°C when alginate, M-enriched blocks, or G-enriched blocks were used as substrates (Fig. S3A). A thermostability assay further showed that the alginate-degrading activity of rAly-6 was stable at 0 to 30°C, and more than 60% activity was retained even if the enzyme was preincubated at 30°C for 24 h (Fig. S3C). The optimal pH, determined at 40°C in both 50 mM sodium acetate-acetic acid (NaAc-HAc) buffer and 50 mM NaH2PO4-Na2HPO4 buffer, was 6.0 (Fig. S3B). The enzyme retained more than 60% of the highest activity after preincubation for 2 h at pH 5.0 to 8.0 (Fig. S3B). The TF-factor-fused recombinant enzyme rTF-Aly-6 showed similar biochemical characteristics, i.e., the optimal temperature and pH value for catalysis and the enzyme’s thermal and pH stabilities, to rAly-6.
The alginate lyase activities of rAly-6 were strongly inhibited by 1.0 or 10 mM Ag+, Cu2+, Hg2+, sodium dodecyl sulfonate (SDS), 10 mM Pb2+, Zn2+, Cr3+, Fe3+, or ethylenediaminetetraacetic acid (EDTA). In contrast, the enzyme activity of rAly-6 was increased to 130 ~ 160% by various concentrations (1.0 or 10 mM) of Co2+, Mn2+, and Ni2+. Chemicals such as glycerol, dithiothreitol (DTT), and the reducing agent β-mercaptoethanol (β-ME) weakly increased the activity of Aly-6 (Fig. S3E). Moreover, the enzyme activity of rAly-6 was strongly increased by increasing the NaCl concentration from 0.0 to 0.5 M, reaching approximately 210% at 0.2 M, whereas it was strongly inhibited by increasing the NaCl concentration from 0.5 to 1.0 M, decreasing to approximately 10% at 1.0 M (Fig. S3D). The results indicated that the alginate lyase Aly-6 is active without NaCl while being adapted to a range of NaCl concentrations, which may be associated with the protein origin, Flammeovirga sp. strain MY04, being derived from coastal environments. Similar to that of rAly-6, the enzyme activities of Avi-rAlgL and Pae-rAlgL were both strongly inhibited by 1.0 or 10 mM SDS or 10 mM Cu2+, Hg2+, Pb2+, Zn2+, Cr3+, or Fe3+ (Fig. S4A and B). Moreover, their enzyme activities were NaCl independent and increased by increasing the NaCl concentration from 0 to 1.0 M, reaching approximately 130% at over 0.2 M NaCl, while Avi-rAlgL activity reached 160% at 0.6 M (Fig. S4C and D).
Under optimal conditions (40°C in 50 mM NaAc-HAc buffer, pH 6.0), the enzyme rAly-6 showed specific activities of 726 ± 2.2, 525 ± 3.5, 196 ± 2.9 U/mg in the degradation of alginate, M blocks, and G blocks. These results demonstrated that the enzyme activity of Aly-6 against M blocks is almost 2.71-fold that against G blocks, and therefore Aly-6 is an M-preferred lyase. Similarly, the reported M-specific lyases Pae-rAlgL and Avi-rAlgL exhibited activities of 2685/4219, 5704/8085, and 144/98 U/mg in the degradation of the corresponding substrates (Table. 1), respectively.
3.4 Polysaccharide degradation patterns and oligosaccharide products.
Unsaturated oligosaccharides with a high degree of poly-Merization (DP) were the main products in the initial stage of the reaction and were then gradually converted into smaller oligosaccharides (Fig. S5A and B), indicating that Avi-rAlgL and Pae-rAlgL are typical endo-type alginate lyases. The final products were similar (UDP2 ~ UDP7) but with differing molar ratios for each component (Fig. S5D). These enzymes also degrade unsaturated oligosaccharides endolytically. Unlike the two endolytic agents, rAly-6 exhibited a different degradation behavior (Fig. S5C). To further identify the structures of oligosaccharides produced by rAly-6, Avi-rAlgL, and Pae-rAlgL, six size-defined oligosaccharide fractions, UDP2 ~ UDP7 (Avi-rAlgL and Pae-rAlgL), were isolated from the final products, and UDP3 ~ UDP6 (rAly-6) were isolated from intermediate products of alginate digestion as described above. The 1H-NMR chemical shifts of the protons of the ∆ unit at the nonreducing end of unsaturated alginate oligosaccharides are strongly affected by the properties of the nearest monosaccharide residues and the structure of the residue next to the ∆ unit (29, 38–40). In the case of UDP3 ~ UDP6 produced by rAly-6, the specific signal at 5.65 ppm of H-4 of ∆G was strong, but no H-4 of ∆M was observed, indicating that rAly-6 (Fig. 2C) is currently the first alginate lyase that produced ∆G-terminal oligosaccharide products.
In addition, the UDP2 ~ UDP7 products from Avi-rAlgL and Pae-rAlgL were also analyzed. The observed characteristic chemical shift value indicated that almost all the UDP2 (5.65 ppm) produced by Avi-rAlgL and Pae-rAlgL was ΔM, while the UDP5 ~ UDP7 fractions produced by Pae-rAlgL and UDP4 ~ UDP7 produced by Avi-rAlgL (5.70 ppm) were ΔG (Fig. 2A and B). The signals at 5.70 ppm and 5.60 ppm of UDP3 (Avi-rAlgL and Pae-rAlgL) and UDP4 (Pae-rAlgL), respectively, showed that UDP3 was ΔM and UDP4 was ΔG, with molar ratios of 100:104, 134:100, and 100:196. In short, the products produced by Avi-rAlgL and Pae-rAlgL during alginate degradation are similar, and the difference is that only UDP3 produced by Avi-rAlgL, not UDP3 and UDP4 from Avi-rAlgL, has two types of nonreducing ends, ∆M and ∆G. Therefore, although the characteristic succession of the nr end structure of the oligosaccharide end products of the two endo-type alginate lyases is that as the degree of poly-Merization increases, the proportion of nonreducing-end-containing ∆M of the oligosaccharide gradually decreases and the ∆G gradually increases to completely replace ∆M, these two enzymes preferred to produce large oligosaccharides (> UDP5) in which the nr end primarily contained ΔG units.
To investigate the oligosaccharide-degrading properties of the three alginate lyases, size-defined saturated oligosaccharides (M2 ~ M5, G2 ~ G5) were used as testing substrates. Pae-rAlgL and Avi-rAlgL degraded saturated poly-M oligosaccharides (M3-M5) but did not degrade M2 or G5 (Fig. 3C and D). When the saturated M5 sugars were degraded, the final products were mainly unsaturated UM2 (disaccharide), with a small amount of unsaturated UM3 (trisaccharide) and unsaturated UM4 (tetrasaccharide). UM2 is mainly produced when saturated M3 is degraded, while UM3 is mainly produced from M4. This indicates that the two enzymes Pae-rAlgL and Avi-rAlgL are M-specific alginate lyases that can react with the smallest saturated oligosaccharide substrate, M3, and that the smallest product is saturated monosaccharide. In contrast, rAly-6 degrades both M and G series oligosaccharide fragments (Fig. 3A and B), although the product absorption intensity (area integral) of M is significantly higher than that of G (approximately 5:1 ~ 10:1). For example, oligosaccharide yield produced by degradation of M5 is 8 ~ 10-fold that of G5. M2 and G2 were not degraded. The products of degradation of M3/G3 are UM2/UG2 and saturated monosaccharides M/G, M4/G4 (UM2/UG2, UM3/UG3, M/G, and M2/G2), and M5/G5 (UM2/UG2, UM3/UG3, M/G and M2/G2). This fully showed that rAly-6 can degrade both M/G-rich oligosaccharide fragments with M preference.
To exactly determine the substrate degradation orientation of Aly-6 and two endolytic lyases, saturated/unsaturated alginate pentasaccharides (DP5) were labeled with 2-AB at the reducing end and further digested with enzymes in a time-course experiment. A gel filtration assay showed that the digestion of 2-AB-M5/2-AB-UDP5 by Aly-6 yielded a series of 2-AB-labeled oligosaccharide products with high molecular masses at the beginning of the reaction, and then, the larger products 2-AB-UM4/2-AB-UDP4 and 2-AB-UM3/2-AB-UDP3 were gradually converted into the final product, 2-AB-UM2/2-AB-UDP2 (Fig. 4A and C). In particular, the final product 2-AB-UM2 was formed in the initial stage of the reaction, and its yield gradually increased with reaction time. For the same experiment with 2-AB-G5, approximately 45% of the substrate was degraded to produce 2-AB-UG2 in equimolar amounts (Fig. 4B). The results indicated that the recombinant alginate lyase rAly-6 cleaved 2-AB-M5/2-AB-G5/2-AB-UDP5 as a monosaccharide-yielding exo-type enzyme; that is, it gradually cleaved one molecule of a saturated M/G monosaccharide and two unsaturated monosaccharide units (∆) from the nonreducing end of the substrate chain until one molecule of 2-AB-UM2/2-AB-UG2 remained.
As shown in Fig. 4D and E, 2-AB-M5 was produced by Pae-rAlgL or Avi-rAlgL, 2-AB-UM4 and 2-AB-UM3 were first produced, and finally 2-AB-UM3 and 2-AB-UM2 were the main products. However, when 2-AB-M4 was degraded, 2-AB-UM3 was the main final product (Fig. S6). In summary, this indicates that when Pae-rAlgL or Avi-rAlgL degrades 2-AB-M5, the main product of 2-AB-UM2 is not produced by continuous exocytosis, which is a variable substrate degradation mode with internal and external mode behavior.
To investigate the degradation patterns of the enzymes reacting against oligosaccharide substrates, each size-defined unsaturated oligosaccharide chain, i.e., the UDP2, UDP3, UDP4, and UDP5 fractions, was reacted with rAly-6 using the same strategy as described previously. After further enzymatic reaction with rAly-6, the final digests of each size-defined oligosaccharide fraction were analyzed via gel filtration HPLC. The results showed that UDP3 and UDP2 were produced by degradation of UDP5 and UDP4, which contained only the ΔG-terminus, and the degradation ratio of UDP3 was approximately 5%, while that of UDP3 was 60% (mix of ΔM/ΔG-termini) (Fig. 5A). Furthermore, characteristic UDP2/M2/G2 fractions were degraded by rAly-6 (Fig. 5B), showing that the unsaturated disaccharide fragment (ΔM or ΔG) was the smallest oligosaccharide substrate of rAly-6, wherein ΔM was more easily degraded than ΔG, but neither could be completely degraded (Table. 2). Based on the above, the recombinant alginate lyase rAly-6 is an M-preferred alginate lyase that can easily degrade alginate chains larger than trisaccharides or alginate chains containing ΔM termini but cannot easily degrade alginate trisaccharide and disaccharide chains, particularly those containing ΔG, GG or MM termini.
3.5 Catalytic mechanism of rAly-6.
Aly-6 was determined to be composed of α6/α6 barrels and antiparallel β-chains as a sheet (Fig. S7A) with a crack-like catalytic cavity using Alg17c (PDB: 4NEI) as a template (Fig. S7B); this fold is significantly different from the barrel or jelly roll folds used by other PL family alginate lyases, with the extra peptide Gly224 ~ Ala248 (Fig. 6A). After the extra peptide Gly224 ~ Ala248 was truncated, there was little change in enzyme activity (Table. 3), indicating that the extra peptide was not a core essential structure and had a weak effect on the enzyme. Mutants H197A and W200A showed 46.09/144.53 U/mg activity on alginate (Table. 3), indicating that HNH197STW200 is the catalytic motif of Aly-6, which is a representative member of the PL17 subfamily. Moreover, H426A, Y269A, and N144A were approximately completely inactivated. According to similar literature reports, the sensitivity of this mutation inactivation is determined by the characteristics of Aly-6 as an exolytic lyase.
Table 1
Activity of alginate lyases
| Alginate (U/mg) | M Block (U/mg) | G Block (U/mg) |
rAly6 | 726 ± 2.2 | 525 ± 3.5 | 196 ± 2.9 |
rTF-Aly6 | 692 | 547.9 | 183 |
rAly6-Lm | 34.6 | 25.1 | 5 |
rAly6-Hpm | 48 | 19.5 | 8 |
rAlgL-Pae | 2685 | 5704 | 144 |
rAlgL-Avi | 4219 | 8085 | 98 |
Table 2
Oligosaccharides degraded by rAly-6
Test Substrate | Product (s) | Degradation ratio (%) |
ΔG | Δ | 5% |
ΔM | Δ | 90% |
GG | --- | --- |
MM | --- | --- |
ΔGX/ΔMX | UDP2 | 60% |
ΔGX | UDP2 | 5% |
ΔGXX | UDP3, UDP2 | 95% |
ΔGXXX | UDP3, UDP2 | 98% |
Table 3
Aly-6 mutant activity analysis
| mutants | Activity (U/mg) |
Metal ions | H426A | 0 |
Extra peptide | G224-A248 | 367 |
NNHSYW200 | W200A | 144.53 |
Active site residues | H197A | 46.09 |
Y231A | 566.4 |
Y269A | 9.709 |
N144A | 16.07 |