**1. Introduction**

Alginate is an acidic heteropolysaccharide comprising two kinds of uronic acid, β-D-mannuronate and α-L-guluronate [1–3]. This polysaccharide exists as a structural material in cell-wall matrices of brown algae and biofilms of certain bacteria. Since alginate solution shows high viscosity and forms an elastic gel upon chelating Ca2+, it has long been used as viscosifier and gelling agent in the fields of food and pharmaceutical industries. Alginate oligosaccharides produced by alginate lyases have also been recognized as functional materials since they exhibit various biological functions; e.g., promotion of root growth in higher plants [4,5], acceleration of growth rate of *Bifidobacterium* sp. [6], and promotion of penicillin production in *Penicillium chrysogenum* [7]. Anti-oxidant [8], anti-coagulant [9], anti-inflammation [10], and anti-infectious disease [11] are also bioactivities of alginate oligosaccharides. Recently, 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH), an end reaction product of alginate lyases, was proven to be available as a carbon source for ethanol fermentation by the genetically modified microbes [12–14]. Furthermore, 2-keto-3-deoxyaldonic acids like 2-keto-3-deoxy-D-gluconate (KDG) and 2-keto-3-deoxy-6-phosphogluconate (KDPG), which are intermediates in alginate metabolism, have been expected as leading compounds for antibiotics, antiviral agents, and other drugs and medicines [15]. Thus, such alginate-derived products are regarded as promising materials in various practical applications.

Alginate-degrading enzymes have been investigated in many organisms such as soil bacteria [16–21], marine bacteria [22–29], marine gastropods [30–33], and seaweeds [3,34]. Endolytic and exolytic alginate lyases split glycosyl linkages of alginate via β-elimination mechanism producing unsaturated oligosaccharides and monosaccharide, where a double bond is introduced between C4 and C5 of the newly formed non-reducing terminus [35]. Unsaturated monosaccharide, the end product of alginate lyases, is spontaneously [20] and/or enzymatically [36] converted to an open chain form, DEH, and further converted to KDG by the NAD(P)H-dependent DEH reductase. The KDG is phosphorylated to KDPG by KDG kinase and then split to pyruvate and glyceraldehyde-3-phosphate (GAP) by KDPG aldolase. The alginate-derived pyruvate and GAP are finally metabolized by Kreb's cycle. Bacterial alginate lyases have been identified in many species, e.g., *Sphingomonas* sp. [16,17], *Flavobacterium* sp. [26,27], *Saccharophagus* sp. [22,23], *Vibrio* sp. [29], and *Pseudomonas* sp. [20,21]. *Sphingomonas* sp. strain A1 possesses four kinds of alginate lyases, A1-I–IV, whose sequential action completely depolymerizes alginate to DEH [16,17]. *Flavobacterium* sp. strain UMI-01 also possesses four kinds of alginate lyases, FlAlyA, FlAlyB, FlAlyC and FlAlex, whose cooperative action efficiently degrades alginate to DEH [27]. Meanwhile, *Saccharophagus degradans* strain 2-40T possesses two kinds of alginate lyases, Alg7D and Alg17C, which degrade alginate to unsaturated disaccharide and DEH [22,23]. The alginate-derived DEH is reduced to KDG by NAD(P)H-dependent DEH reductases as described above. Recently, this enzyme was identified in *Sphingomonas* sp. strain A1 [18,19], *Flavobacterium* sp. strain UMI-01 [28], *S. degradans* strain 2-40<sup>T</sup> [24], *Vibrio splendidus* 12B01 [13], and marine gastropod *Haliotis discus hannai* [37]. The bacterial DEH reductases were classified under short-chain dehydrogenases/reductases (SDR) superfamily, while the gastropod enzyme was identified as a member of the aldo-keto reductase (AKR) superfamily. Information about alginate lyases and DEH reductases has been continuously accumulated; however, KDG kinase and KDPG aldolase have not been so well investigated.

Under these circumstances, DEH reductase, KDG kinase, and KDPG aldolase were recently characterized in *S. degradans* 2-40T, a member of the phylum *Proteobacteria* [25]. The combined action of these enzymes could convert DEH to pyruvate and GAP in vitro. On the other hand, we also found the existence of alginate-assimilating gene cluster in the genome of *Flavobacterium* sp. strain UMI-01, a member of the phylum *Bacteroidetes* [27,28]. The endolytic and exolytic alginate lyase genes, *flalyA* and *flalyB*, and a DEH reductase gene, *flred*, are located in operon A, and KDG kinase-like gene *flkin* (GenBank accession number, BAQ25538) and KDPG aldolase-like gene *flald* (GenBank accession number, BAQ25539) are in operon B (Figure 1). The alginate lyases and DEH reductase of this bacterium have already been characterized [26–28]; however, KDG kinase and KDPG aldolase have not been identified yet. The amino acid sequences deduced from *flkin* and *flald* showed only 19% and 22% identities, respectively, with those of the corresponding enzymes from *S. degradans* 2-40<sup>T</sup> [25]. These low sequence identities suggest that the properties of *Flavobacterium* (*Bacteroidetes*) enzymes may be somewhat different from those of *Saccharophagus* (*Proteobacteria*) enzymes. Therefore, in the present

study, we first characterized the primary structures of KDG kinase and KDPG aldolase, FlKin and FlAld, of the strain UMI-01 compared with those of other bacterial enzymes. Then, we investigated enzymatic properties of proteins encoded by *flkin* and *flald* using recombinant enzymes, recFlKin and recFlAld. Furthermore, we constructed an in vitro alginate-metabolizing system using recFlKin and recFlAld, along with recombinant alginate lyases and DEH reductase of this bacterium to confirm that this enzyme system can produce pyruvate and GAP from alginate in vitro.

**Figure 1.** Alginate-assimilating enzyme genes in the genome of *Flavobacterium* sp. strain UMI-01. Yellow, alginate-lyase genes; pink, KdgF-like protein gene; white, transcriptional regulator-like protein genes; gray, membrane transporter-like genes; orange, 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) reductase gene; red, 2-keto-3-deoxy-D-gluconate (KDG) kinase-like gene and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase-like gene. Arrows P1 and P2 and arrows T1 and T2 indicate predicted promoters and terminators, respectively.

#### **2. Results**

#### *2.1. Characteristics in the Primary Structures of FlKin and FlAld*

Deduced amino acid sequences of *flkin* and *flald* were compared with those of KDG kinases and KDPG aldolases from several *Proteobacteria* and *Bacteroidetes* species. Enzymes from two *Archaea* species are also included in the comparison of KDG kinases. FlKin showed considerably low amino acid identity (15%–26%) with KDG kinases from *Proteobacteria* species, i.e., *Escherichia coli* (GenBank accession number, WP\_024175791) [38], *Serratia marcescens* (GenBank accession number, ABB04497) [39], and *S. degradans* 2-40T (GenBank accession number, ABD82535) [25], and archaea, i.e., *Sulfolobus solfataricus* (GenBank accession number, WP\_009991690) [40–42] and *Thermus thermophiles* (GenBank accession number, WP\_011229211) [43] (Figure 2). Meanwhile, the sequence of FlKin showed relatively high identities (47%–68%) with the enzymes from *Bacteroidetes* species, i.e., *Gramella forsetii* KT0803 (GenBank accession number, CAL66135), *Dokdonia* sp. MED134 (GenBank accession number, WP\_016501275), and *Lacinutrix* sp. 5H-3-7-4 (GenBank accession number, AEH01605). However, substrate-recognition residues of KDG kinase, which were identified in the *S. solfataricus* enzymes [42], i.e., Gly34, Tyr90, Tyr106, Arg108, Arg166, Asp258, and Asp294, were entirely conserved in FlKin as Gly34, Tyr89, Tyr104, Arg106, Arg169, Asp280, and Asp317, respectively. FlAld also showed low amino acid identity (22%–25%) with KDPG aldolases from *Proteobacteria* species such as *E. coli* (GenBank accession number, WP\_000800517) [44,45], *Zymomonas mobilis* (GenBank accession number, S18559) [44], *Pseudomonas putida* (GenBank accession number, WP\_016501275) [44,46], and *S. degradans* 2-40<sup>T</sup> (GenBank accession number, ABD80644) [25] (Figure 3). Meanwhile, the sequence identities between FlAld and enzymes from other *Bacteroidetes* species such as *G. forsetii* KT0803 (GenBank accession number, KT0803), *Dokdonia* sp. MED134 (GenBank accession number, WP\_013749799), and *Lacinutrix* sp. 5H-3-7-4 (GenBank accession number, AEH01606) were 61%–65%. Catalytic residue Lys133 and substrate-recognition residues, Glu45, Arg49, Thr73, Pro94 and Phe135 identified in the *E. coli* enzyme [45], were conserved in FlAld except for the substitution of Thr73 by Ser. Phylogenetic analyses for KDG kinases and KDPG aldolases (Figure 4A,B) suggested that the *Bacteroidetes* enzymes

are somewhat phylogenetically deviated from the *Proteobacteria* (and *Archaea*) enzymes. Therefore, we decided to examine if FlKin and FlAld of the strain UMI-01 actually possess KDG kinase and KDPG aldolase activities.

**Figure 2.** Multiple alignment for amino acid sequences of FlKin and other KDG kinases. Closed triangles indicate substrate-recognition residues of KDG kinase from *Sulfolobus solfataricus* [42]. FlKin, KDG kinase from *Flavobacterium* sp. strain UMI-01 (GenBank accession number, BAQ25538); Lacin, KDG kinase-like protein from *Lacinutrix* sp. 5H-3-7-4 (GenBank accession number, AEH01605); Dokdo, KDG kinase-like protein from *Dokdonia* sp. MED134 (GenBank accession number, WP\_013749800); Grame, KDG kinase-like protein from *Gramella forsetii* KT0803 (GenBank accession number, CAL66135); Sacch, KDG kinase from *Saccharophagus degradans* 2-40T (GenBank accession number, ABD82535) [25]; Esche, KDG kinase from *Escherichia coli* (GenBank accession number, WP\_024175791) [38]; Serra, KDG kinase from *Serratia marcescens* (GenBank accession number, ABB04497) [39]; Sulfo, KDG kinase from *Sulfolobus solfataricus* (GenBank accession number, WP\_009991690) [40–42]; Therm, KDG kinase from *Thermus thermophiles* (GenBank accession number, WP\_011229211) [43].

**Figure 3.** Multiple alignment for amino acid sequences of FlAld and other KDPG aldolases. Gray box and closed triangles indicate catalytic and substrate-recognition residues of KDPG aldolase from *E. coli* [44,45], respectively. FlAld, KDPG aldolase from *Flavobacterium* sp. strain UMI-01 (GenBank accession number, BAQ25539); Lacin, KDPG aldolase-like protein from *Lacinutrix* sp. 5H-3-7-4 (GenBank accession number, AEH01606); Dokdo, KDPG aldolase-like protein from *Dokdonia* sp. MED134 (GenBank accession number, WP\_013749799); Grame, KDPG aldolase-like protein from *G. forsetii* KT0803 (GenBank accession number, CAL66136); Sacch, KDPG aldolase from *S. degradans* 2-40T (GenBank accession number, ABD80644) [25]; Esche, KDPG aldolase from *E. coli* (GenBank accession number, WP\_000800517) [44,45]; Zymom, KDPG aldolase from *Zymomonas mobilis* (GenBank accession number, S18559) [44]; Pseud, KDPG aldolase from *Pseudomonas putida* (GenBank accession number, WP\_016501275) [44,46].

**Figure 4.** Phylogenetic trees for KDG kinases and KDPG aldolases. Phylogenetic analyses were carried out using amino acid sequences of KDG kinases from *Proteobacteria*, *Archaea* and *Bacteroidetes* species (**A**) and KDPG aldolases from *Proteobacteria* and *Bacteroidetes* species (**B**). Amino acid sequences of KDG kinases and KDPG aldolases were retrieved from the draft or complete genome data deposited in GenBank. Accession numbers for enzyme sequences along with the bacterial species are indicated in the right of each branch. Bootstrap values above 50% are indicated on the root of branches. Scale bar indicates 0.20 amino acid substitution.

#### *2.2. Production of recFlKin and recFlAld, and Their Reaction Products*

Coding regions of *flkin* and *flald* were amplified by PCR with specific primers listed in Table 1, cloned into pCold vector and expressed in *E. coli* BL21 (DE3). The recombinant enzymes were purified by Ni-NTA affinity chromatography. Molecular masses of recFlKin and recFlAld estimated by SDS-PAGE were 39 kDa and 26 kDa, respectively (Figure 5). These values were consistent with the calculated molecular masses of these enzymes, i.e., 39,391 Da and 25,808 Da, which include 8 × Gly + 8 × His-tag [26].

**Figure 5.** SDS-PAGE for recFlKin and recFlAld. Recombinant enzymes were purified Ni-NTA affinity chromatography and subjected to 0.1% SDS–10% polyacrylamide-gel electrophoresis. Proteins in the gel were stained by Coomassie Brilliant Blue R-250. Marker, molecular weight markers (Protein Ladder Broad Range, New England Biolabs, Ipswich, MA, USA).

**Table 1.** Primers used for amplification of *flkin* and *flald* genes.


The recFlKin was allowed to react with KDG in the presence of ATP. TLC analysis suggested that the reaction product was KDPG (Figure 6A). Then, the molecular mass of the reaction product was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF) mass spectrometry (Figure 7A,B). The 257 *m*/*z* peak was considered to be that of KDPG (MW = 258), and the 279 *m*/*z* peak was considered to be that of a sodium-salt form of KDPG. These results indicate that the reaction product of recFlKin is KDPG. Thus, we concluded that the protein encoded by *flkin* is KDG kinase. Here, it should be noted that the peak intensities of KDPG were considerably low. This was ascribable to the low ionization level of KDPG. Therefore, we attempted to improve the signal intensity of KDPG using other matrices, e.g., 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid. Unfortunately, signal intensity of KDPG was not improved much. We still need to investigate the suitable conditions for the detection of KDPG.

Reaction products of recFlAld were also analyzed by TLC (Figure 6B). recFlAld produced two kinds of reaction products with different mobility on TLC. According to their mobility, they were regarded as pyruvate and GAP. The staining intensity of pyruvate was significantly low compared with that of GAP. This difference was ascribable to the difference in the reactivity between pyruvate and GAP with 2,4-dinitrophenylhydrazine (DNP). Namely, GAP showed much higher reactivity with DNP than pyruvate. Then, the reaction products of recFlAld were subjected to MALDI-TOF mass spectrometry. The 87 *m*/*z* and 169 *m*/*z* peaks corresponding to pyruvate (MW = 88) and GAP

(MW = 170), respectively, were observed. The peak intensity of GAP was small (Figure 7C,D). This appeared to be due to the decomposition of GAP during the mass spectrometric analysis. Thus, we may conclude that recFlAld is the KDPG aldolase that splits KDPG to pyruvate and GAP.

**Figure 6.** Thin-layer chromatography (TLC) analyses for reaction products of recFlKin and recFlAld. (**A**) Reaction products produced by recFlKin. The reaction products were visualized by spraying 10% (*v*/*v*) sulfuric acid in ethanol followed by heating at 130 ◦C for 10 min. M, standard KDPG; (**B**) Reaction products of recFlAld. The reaction products were visualized with 0.5% (*w*/*v*) 2,4-dinitrophenylhydrazine (DNP)–20% (*v*/*v*) sulfuric acid. The color was graphically inverted to ease the recognition of spots. M1, standard pyruvate; M2, standard glyceraldehyde-3-phosphate (GAP). Stained materials near the original position are GAP oligomers.

**Figure 7.** Mass spectrometry for reaction products of recFlKin and recFlAld. The reaction products prepared as in Section 4.10 were subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF) mass spectrometry, and analyzed by negative-ion mode. (**A**,**B**) KDG before and after the recFlKin reaction, respectively; (**C**,**D**) KDPG before and after the recFlAld reaction, respectively. Reaction products are indicated with red letters along with molecular masses above the peaks.
