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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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Analysis for the reaction products of DEH produced by recFlRed. The reaction was performed at 25 °C in a reaction mixture consisting of 10 mM DEH, 10 mM NADH, 10 mM potassium phosphate buffer (pH 7.0), 100 mM KCl, and 2.5 μg/mL recFlRed for 0–90 min. (A) Thin-layer chromatography for the products stained with 10% sulfuric acid in ethanol. Di- and Tri- indicate the standard disaccharide and trisaccharide produced by FlAlyA [20], respectively; (B) Thin-layer chromatography for the products stained with 4.5% thiobarbituric acid; (C) Mass spectrogram of DEH (m/z 175); (D) Mass spectrogram of the reaction product indicating the production of KDG (m/z 177).
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marinedrugs-13-00493-f004: Analysis for the reaction products of DEH produced by recFlRed. The reaction was performed at 25 °C in a reaction mixture consisting of 10 mM DEH, 10 mM NADH, 10 mM potassium phosphate buffer (pH 7.0), 100 mM KCl, and 2.5 μg/mL recFlRed for 0–90 min. (A) Thin-layer chromatography for the products stained with 10% sulfuric acid in ethanol. Di- and Tri- indicate the standard disaccharide and trisaccharide produced by FlAlyA [20], respectively; (B) Thin-layer chromatography for the products stained with 4.5% thiobarbituric acid; (C) Mass spectrogram of DEH (m/z 175); (D) Mass spectrogram of the reaction product indicating the production of KDG (m/z 177).

Mentions: The reaction products produced by recFlRed were then analyzed by thin-layer chromatography (TLC) and mass spectroscopy. As shown in Figure 4A, DEH was hardly detected on TLC by the sulfuric-acid detection; however, a clear band corresponding to KDG appeared in the reaction time at 30 and 90 min. Conversion of DEH to KDG was more definitely detected by thiobarbituric acid detection (Figure 4B). Namely, the original DEH band was gradually decreased with the extension of reaction time, while a KDG band with the mobility smaller than DEH concomitantly appeared. These results suggested that the DEH was converted to KDG by the action of recFlRed. The molecular masses of the DEH and the reaction product KDG were subsequently determined by MALDI-TOF-MS (Figure 4C,D). The peak with 175 m/z corresponding to DEH was detected before the reaction (Figure 4C), while this peak decreased by the reaction and instead a new peak with 177 m/z corresponding to KDG appeared (Figure 4D). These results supported the conversion of DEH to KDG by the action of recFlRed.


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)

Analysis for the reaction products of DEH produced by recFlRed. The reaction was performed at 25 °C in a reaction mixture consisting of 10 mM DEH, 10 mM NADH, 10 mM potassium phosphate buffer (pH 7.0), 100 mM KCl, and 2.5 μg/mL recFlRed for 0–90 min. (A) Thin-layer chromatography for the products stained with 10% sulfuric acid in ethanol. Di- and Tri- indicate the standard disaccharide and trisaccharide produced by FlAlyA [20], respectively; (B) Thin-layer chromatography for the products stained with 4.5% thiobarbituric acid; (C) Mass spectrogram of DEH (m/z 175); (D) Mass spectrogram of the reaction product indicating the production of KDG (m/z 177).
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Related In: Results  -  Collection

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

marinedrugs-13-00493-f004: Analysis for the reaction products of DEH produced by recFlRed. The reaction was performed at 25 °C in a reaction mixture consisting of 10 mM DEH, 10 mM NADH, 10 mM potassium phosphate buffer (pH 7.0), 100 mM KCl, and 2.5 μg/mL recFlRed for 0–90 min. (A) Thin-layer chromatography for the products stained with 10% sulfuric acid in ethanol. Di- and Tri- indicate the standard disaccharide and trisaccharide produced by FlAlyA [20], respectively; (B) Thin-layer chromatography for the products stained with 4.5% thiobarbituric acid; (C) Mass spectrogram of DEH (m/z 175); (D) Mass spectrogram of the reaction product indicating the production of KDG (m/z 177).
Mentions: The reaction products produced by recFlRed were then analyzed by thin-layer chromatography (TLC) and mass spectroscopy. As shown in Figure 4A, DEH was hardly detected on TLC by the sulfuric-acid detection; however, a clear band corresponding to KDG appeared in the reaction time at 30 and 90 min. Conversion of DEH to KDG was more definitely detected by thiobarbituric acid detection (Figure 4B). Namely, the original DEH band was gradually decreased with the extension of reaction time, while a KDG band with the mobility smaller than DEH concomitantly appeared. These results suggested that the DEH was converted to KDG by the action of recFlRed. The molecular masses of the DEH and the reaction product KDG were subsequently determined by MALDI-TOF-MS (Figure 4C,D). The peak with 175 m/z corresponding to DEH was detected before the reaction (Figure 4C), while this peak decreased by the reaction and instead a new peak with 177 m/z corresponding to KDG appeared (Figure 4D). These results supported the conversion of DEH to KDG by the action of recFlRed.

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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Related in: MedlinePlus