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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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Nucleotide and deduced amino-acid sequences of flred and the structure of pCold I vector. (A) Nucleotide and deduced amino-acid sequences of flred; (B) Structure of pCold I vector for the expression of recFlRed.
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marinedrugs-13-00493-f005: Nucleotide and deduced amino-acid sequences of flred and the structure of pCold I vector. (A) Nucleotide and deduced amino-acid sequences of flred; (B) Structure of pCold I vector for the expression of recFlRed.

Mentions: Coding region of flred gene was amplified by genomic PCR using specific forward and reverse primers, 5′-GTAGTAAATAATTAATTTAGAATAAAG-3′ and 5′-AGTTTAATCAAGGACAAAAAAGGCGTC-3′, respectively, which were synthesized on the basis of 5′- and 3′-flanking sequences of flred (see Figure 1). PCR was performed in 50 μL of reaction mixture containing 10 ng of total DNA and 1 µM each primer using Phusion® Hot Start Flex 2× Master Mix (New England Biolabs, Ipswich, MA, USA). After preheating at 98 °C for 2 min, the reaction of 98 °C for 10 s, 50 °C for 15 s, and 72 °C for 30 s was repeated 30 cycles. The PCR product was treated with A-attachment mix (Toyobo, Osaka, Japan) and ligated to pTac-1. The nucleotide sequence of the cloned DNA in pTac-1 was analyzed with a BigDye-Terminator Cycle Sequence kit (Applied Biosystems, Foster City, CA, USA) and a DNA sequencer 3130xl (Applied Biosystems, Foster, CA, USA). Restriction sites were introduced to the 5′- and 3′-termini of the cloned flred using the primers, 5′-AGGTAATACACCATGGGTAATTTAAATGAAAAAGTTG-3′ (Nco I site was underlined) and 5′-CACCTCCACCGGATCCTATTCCTAAAGCTTGACCTCC-3′ (BamH I site was underlined). The amplified DNA was ligated to pCold vector digested by Nco I and BamH I using In-Fusion system (Clontech Laboratories, Mountain View, CA, USA). The pCold I vector had been modified to add 8×Gly+8×His-tag to the C-terminus of recombinant protein and to remove original N-terminal translation enhancing element, His-tag and Factor Xa recognition site as reported previously [20]. The nucleotide and deduced amino-acid sequences of flred and the circular map for the expression pCold I vector are shown in Figure 5A,B. The resultant plasmid was introduced to E. coli BL21 (DE3), and it was cultured at 37 °C overnight in 2xYT medium. To express recombinant FlRed (recFlRed), 0.1 mM IPTG was added to the culture and incubated at 15 °C for 12 h. Bacterial cells were harvested by centrifugation at 5000× g for 15 min and homogenized by sonication in a buffer containing 10 mM imidazole-HCl (pH 8.0), 0.5 M NaCl, 1% Triton X-100, and 0.01 mg/mL lysozyme. The homogenate was centrifuged at 10,000× g for 15 min, and recFlRed in the supernatant was adsorbed to Ni-NTA resin in a conical tube with gentle mixing. After the incubation for 30 min on ice, resin was set in a disposable plastic column and washed with a buffer containing 30 mM imidazole-HCl (pH 8.0)–0.5 M NaCl. recFlRed was eluted with 150 mM imidazole-HCl (pH 8.0)–0.5 M NaCl from the column and dialyzed against 0.1 M NaCl–10 mM sodium phosphate buffer (pH 7.5) before use.


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)

Nucleotide and deduced amino-acid sequences of flred and the structure of pCold I vector. (A) Nucleotide and deduced amino-acid sequences of flred; (B) Structure of pCold I vector for the expression of recFlRed.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4306948&req=5

marinedrugs-13-00493-f005: Nucleotide and deduced amino-acid sequences of flred and the structure of pCold I vector. (A) Nucleotide and deduced amino-acid sequences of flred; (B) Structure of pCold I vector for the expression of recFlRed.
Mentions: Coding region of flred gene was amplified by genomic PCR using specific forward and reverse primers, 5′-GTAGTAAATAATTAATTTAGAATAAAG-3′ and 5′-AGTTTAATCAAGGACAAAAAAGGCGTC-3′, respectively, which were synthesized on the basis of 5′- and 3′-flanking sequences of flred (see Figure 1). PCR was performed in 50 μL of reaction mixture containing 10 ng of total DNA and 1 µM each primer using Phusion® Hot Start Flex 2× Master Mix (New England Biolabs, Ipswich, MA, USA). After preheating at 98 °C for 2 min, the reaction of 98 °C for 10 s, 50 °C for 15 s, and 72 °C for 30 s was repeated 30 cycles. The PCR product was treated with A-attachment mix (Toyobo, Osaka, Japan) and ligated to pTac-1. The nucleotide sequence of the cloned DNA in pTac-1 was analyzed with a BigDye-Terminator Cycle Sequence kit (Applied Biosystems, Foster City, CA, USA) and a DNA sequencer 3130xl (Applied Biosystems, Foster, CA, USA). Restriction sites were introduced to the 5′- and 3′-termini of the cloned flred using the primers, 5′-AGGTAATACACCATGGGTAATTTAAATGAAAAAGTTG-3′ (Nco I site was underlined) and 5′-CACCTCCACCGGATCCTATTCCTAAAGCTTGACCTCC-3′ (BamH I site was underlined). The amplified DNA was ligated to pCold vector digested by Nco I and BamH I using In-Fusion system (Clontech Laboratories, Mountain View, CA, USA). The pCold I vector had been modified to add 8×Gly+8×His-tag to the C-terminus of recombinant protein and to remove original N-terminal translation enhancing element, His-tag and Factor Xa recognition site as reported previously [20]. The nucleotide and deduced amino-acid sequences of flred and the circular map for the expression pCold I vector are shown in Figure 5A,B. The resultant plasmid was introduced to E. coli BL21 (DE3), and it was cultured at 37 °C overnight in 2xYT medium. To express recombinant FlRed (recFlRed), 0.1 mM IPTG was added to the culture and incubated at 15 °C for 12 h. Bacterial cells were harvested by centrifugation at 5000× g for 15 min and homogenized by sonication in a buffer containing 10 mM imidazole-HCl (pH 8.0), 0.5 M NaCl, 1% Triton X-100, and 0.01 mg/mL lysozyme. The homogenate was centrifuged at 10,000× g for 15 min, and recFlRed in the supernatant was adsorbed to Ni-NTA resin in a conical tube with gentle mixing. After the incubation for 30 min on ice, resin was set in a disposable plastic column and washed with a buffer containing 30 mM imidazole-HCl (pH 8.0)–0.5 M NaCl. recFlRed was eluted with 150 mM imidazole-HCl (pH 8.0)–0.5 M NaCl from the column and dialyzed against 0.1 M NaCl–10 mM sodium phosphate buffer (pH 7.5) before use.

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