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Identification of a 4-deoxy-L-erythro-5-hexoseulose uronic acid reductase, FlRed, in an alginolytic bacterium Flavobacterium sp. strain UMI-01.

Inoue A, Nishiyama R, Mochizuki S, Ojima T - Mar Drugs (2015)

Bottom Line: Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp.As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH.On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Marine Biotechnology and Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan. inouea21@fish.hokudai.ac.jp.

ABSTRACT
In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11). The monosaccharide is non-enzymatically converted to 4-deoxy-L-ery thro-5-hexoseulose uronic acid (DEH), then reduced to 2-keto-3-deoxy-D-gluconate (KDG) by a specific reductase, and metabolized through the Entner-Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%-88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.

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Comparison of amino-acid sequences between FlRed and other SDR-superfamily enzymes. Open circles show the residues conserved in the Rossmann fold cofactor-binding motif Thr-Gly-x-x-x-Gly-x-Gly. Closed circles represent the residues configuring catalytic tetrad. Gray box indicates the position corresponding to Arg39 of A1-R, which is responsible for NADPH specificity of A1-R. Blue and red lines represent the positions of β-sheets (S1–S7) and α-helices (H1–H10) in A1-R [18]. FlRed, DEH reductase from Flavobacterium sp. UMI-01 (present study); Formosa, acetoin (diacetyl) reductase from Formosa agariphila KMM 3901 (GenBank accession number, CDF80389); Flavobacterium, short-chain dehydrogenase from Flavobacterium frigidarium (GenBank accession number, WP_026707247); Cytophaga, short-chain dehydrogenase from Cytophaga fermentans (GenBank accession number, WP_027472031); Lewinella, short-chain dehydrogenase from Lewinella cohaerens (GenBank accession number, WP_020539555); Cellulophaga, short-chain dehydrogenase from Cellulophaga algicola (GenBank accession number WP_013552902); Zobellia, short-chain dehydrogenase from Zobellia galactanivorans (GenBank accession number, WP_013993961); A1-R, NADPH-dependent DEH reductase A1-R from Sphingomonas sp. A1 [18].
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marinedrugs-13-00493-f002: Comparison of amino-acid sequences between FlRed and other SDR-superfamily enzymes. Open circles show the residues conserved in the Rossmann fold cofactor-binding motif Thr-Gly-x-x-x-Gly-x-Gly. Closed circles represent the residues configuring catalytic tetrad. Gray box indicates the position corresponding to Arg39 of A1-R, which is responsible for NADPH specificity of A1-R. Blue and red lines represent the positions of β-sheets (S1–S7) and α-helices (H1–H10) in A1-R [18]. FlRed, DEH reductase from Flavobacterium sp. UMI-01 (present study); Formosa, acetoin (diacetyl) reductase from Formosa agariphila KMM 3901 (GenBank accession number, CDF80389); Flavobacterium, short-chain dehydrogenase from Flavobacterium frigidarium (GenBank accession number, WP_026707247); Cytophaga, short-chain dehydrogenase from Cytophaga fermentans (GenBank accession number, WP_027472031); Lewinella, short-chain dehydrogenase from Lewinella cohaerens (GenBank accession number, WP_020539555); Cellulophaga, short-chain dehydrogenase from Cellulophaga algicola (GenBank accession number WP_013552902); Zobellia, short-chain dehydrogenase from Zobellia galactanivorans (GenBank accession number, WP_013993961); A1-R, NADPH-dependent DEH reductase A1-R from Sphingomonas sp. A1 [18].

Mentions: Figure 1 represents the schematic structure for an alginolytic gene cluster of 15.6 kbp found in strain UMI-01 genome (the nucleotide and deduced amino-acid sequences of individual genes are available from DDBJ, GenBank and EMBL with following accession numbers; FlAlyB, LC005508; KdgF-like protein, LC005509; FlAlyA [20], AB898059; GntR-like protein1, LC005510; sugar permiase, LC005511; SDR-like enzyme, LC005512; Kdg kinase, LC005513; KDPG aldolase, LC005514; SucC-like protein; LC005515; SusD-like protein, LC005516; GntR-like protein2, LC005517). The gene cluster comprised two putative operons, i.e., Op-A and Op-B, which were defined by the occurrence of two sets of promoter and terminator sequences, i.e., P1-T1 and P2-T2, which were predicted according to the consensus sequences proposed by Chen et al. [21,22]. Op-A comprised two alginate lyase genes (FlAlyA and FlAlyB genes), a KdgF-like protein gene, a GntR-like gene, a sugar permiase gene, and an SDR-like enzyme (FlRed) gene (flred), while Op-B comprised a Kdg-kinase gene, a KDPG-aldolase gene, a susC-like protein gene, a susD-like protein gene, and a GntR-like gene. Among these genes, the FlAlyA gene had been identified as the gene encoding the endolytic alginate lyase FlAlyA that belongs to polysaccharide-lyase family 7 (PL-7) [20]. KdgF-like protein gene was suggested to relate to pectin metabolism [23]; however, in this bacterium, it may participate in alginate metabolism. The SDR-like enzyme (FlRed) gene flred in Op-A was considered to be the DEH-reductase gene because of its involvement in the alginolytic operon. We then subjected the deduced amino-acid sequence of FlRed gene to BLAST search and retrieved some sequences of SDR-superfamily enzymes (Figure 2). The amino-acid identity between FlRed and the DEH reductase A1-R [18] was 34%, while the identities between FlRed and other SDR enzymes were 80%–88%. Formosaagariphila [24], Zobellia galactanivorans [25] and Sphingomonas sp. A1 [18] are known as alginate-assimilating bacteria, while Cellulophaga algicola [26], Cytophaga fermentans [27] Flavobacterium frigidarium (GenBank accession number, WP_026707247) and Lewinella cohaerens (GenBank accession number, WP_020539555) have not been identified as alginolytic bacteria. Specific sequence motifs of SDR-family enzymes, i.e., the catalytic tetrad Asn-Tyr-Lys-Ser [28], the cofactor-binding sequence motif Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold were entirely conserved in FlRed as well as other SDR-superfamily enzymes, whereas, a basic amino-acid residue responsible for NADPH-specificity of Sphingomonas A1-R [18], i.e., Arg39, was replaced by acidic residue Glu or Asp in FlRed and other SDR-superfamily enzymes. This suggested that FlRed was not NADPH-dependent but NADH-dependent unlike Sphingomonas A1-R.


Identification of a 4-deoxy-L-erythro-5-hexoseulose uronic acid reductase, FlRed, in an alginolytic bacterium Flavobacterium sp. strain UMI-01.

Inoue A, Nishiyama R, Mochizuki S, Ojima T - Mar Drugs (2015)

Comparison of amino-acid sequences between FlRed and other SDR-superfamily enzymes. Open circles show the residues conserved in the Rossmann fold cofactor-binding motif Thr-Gly-x-x-x-Gly-x-Gly. Closed circles represent the residues configuring catalytic tetrad. Gray box indicates the position corresponding to Arg39 of A1-R, which is responsible for NADPH specificity of A1-R. Blue and red lines represent the positions of β-sheets (S1–S7) and α-helices (H1–H10) in A1-R [18]. FlRed, DEH reductase from Flavobacterium sp. UMI-01 (present study); Formosa, acetoin (diacetyl) reductase from Formosa agariphila KMM 3901 (GenBank accession number, CDF80389); Flavobacterium, short-chain dehydrogenase from Flavobacterium frigidarium (GenBank accession number, WP_026707247); Cytophaga, short-chain dehydrogenase from Cytophaga fermentans (GenBank accession number, WP_027472031); Lewinella, short-chain dehydrogenase from Lewinella cohaerens (GenBank accession number, WP_020539555); Cellulophaga, short-chain dehydrogenase from Cellulophaga algicola (GenBank accession number WP_013552902); Zobellia, short-chain dehydrogenase from Zobellia galactanivorans (GenBank accession number, WP_013993961); A1-R, NADPH-dependent DEH reductase A1-R from Sphingomonas sp. A1 [18].
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4306948&req=5

marinedrugs-13-00493-f002: Comparison of amino-acid sequences between FlRed and other SDR-superfamily enzymes. Open circles show the residues conserved in the Rossmann fold cofactor-binding motif Thr-Gly-x-x-x-Gly-x-Gly. Closed circles represent the residues configuring catalytic tetrad. Gray box indicates the position corresponding to Arg39 of A1-R, which is responsible for NADPH specificity of A1-R. Blue and red lines represent the positions of β-sheets (S1–S7) and α-helices (H1–H10) in A1-R [18]. FlRed, DEH reductase from Flavobacterium sp. UMI-01 (present study); Formosa, acetoin (diacetyl) reductase from Formosa agariphila KMM 3901 (GenBank accession number, CDF80389); Flavobacterium, short-chain dehydrogenase from Flavobacterium frigidarium (GenBank accession number, WP_026707247); Cytophaga, short-chain dehydrogenase from Cytophaga fermentans (GenBank accession number, WP_027472031); Lewinella, short-chain dehydrogenase from Lewinella cohaerens (GenBank accession number, WP_020539555); Cellulophaga, short-chain dehydrogenase from Cellulophaga algicola (GenBank accession number WP_013552902); Zobellia, short-chain dehydrogenase from Zobellia galactanivorans (GenBank accession number, WP_013993961); A1-R, NADPH-dependent DEH reductase A1-R from Sphingomonas sp. A1 [18].
Mentions: Figure 1 represents the schematic structure for an alginolytic gene cluster of 15.6 kbp found in strain UMI-01 genome (the nucleotide and deduced amino-acid sequences of individual genes are available from DDBJ, GenBank and EMBL with following accession numbers; FlAlyB, LC005508; KdgF-like protein, LC005509; FlAlyA [20], AB898059; GntR-like protein1, LC005510; sugar permiase, LC005511; SDR-like enzyme, LC005512; Kdg kinase, LC005513; KDPG aldolase, LC005514; SucC-like protein; LC005515; SusD-like protein, LC005516; GntR-like protein2, LC005517). The gene cluster comprised two putative operons, i.e., Op-A and Op-B, which were defined by the occurrence of two sets of promoter and terminator sequences, i.e., P1-T1 and P2-T2, which were predicted according to the consensus sequences proposed by Chen et al. [21,22]. Op-A comprised two alginate lyase genes (FlAlyA and FlAlyB genes), a KdgF-like protein gene, a GntR-like gene, a sugar permiase gene, and an SDR-like enzyme (FlRed) gene (flred), while Op-B comprised a Kdg-kinase gene, a KDPG-aldolase gene, a susC-like protein gene, a susD-like protein gene, and a GntR-like gene. Among these genes, the FlAlyA gene had been identified as the gene encoding the endolytic alginate lyase FlAlyA that belongs to polysaccharide-lyase family 7 (PL-7) [20]. KdgF-like protein gene was suggested to relate to pectin metabolism [23]; however, in this bacterium, it may participate in alginate metabolism. The SDR-like enzyme (FlRed) gene flred in Op-A was considered to be the DEH-reductase gene because of its involvement in the alginolytic operon. We then subjected the deduced amino-acid sequence of FlRed gene to BLAST search and retrieved some sequences of SDR-superfamily enzymes (Figure 2). The amino-acid identity between FlRed and the DEH reductase A1-R [18] was 34%, while the identities between FlRed and other SDR enzymes were 80%–88%. Formosaagariphila [24], Zobellia galactanivorans [25] and Sphingomonas sp. A1 [18] are known as alginate-assimilating bacteria, while Cellulophaga algicola [26], Cytophaga fermentans [27] Flavobacterium frigidarium (GenBank accession number, WP_026707247) and Lewinella cohaerens (GenBank accession number, WP_020539555) have not been identified as alginolytic bacteria. Specific sequence motifs of SDR-family enzymes, i.e., the catalytic tetrad Asn-Tyr-Lys-Ser [28], the cofactor-binding sequence motif Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold were entirely conserved in FlRed as well as other SDR-superfamily enzymes, whereas, a basic amino-acid residue responsible for NADPH-specificity of Sphingomonas A1-R [18], i.e., Arg39, was replaced by acidic residue Glu or Asp in FlRed and other SDR-superfamily enzymes. This suggested that FlRed was not NADPH-dependent but NADH-dependent unlike Sphingomonas A1-R.

Bottom Line: Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp.As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH.On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Marine Biotechnology and Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan. inouea21@fish.hokudai.ac.jp.

ABSTRACT
In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11). The monosaccharide is non-enzymatically converted to 4-deoxy-L-ery thro-5-hexoseulose uronic acid (DEH), then reduced to 2-keto-3-deoxy-D-gluconate (KDG) by a specific reductase, and metabolized through the Entner-Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%-88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.

Show MeSH
Related in: MedlinePlus