Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of pH and temperature on the activity of recFlRed. (A) SDS-polyacrylamide gel electrophoresis for recFlRed. Mk, marker proteins; (B) pH dependence of recFlRed. Activity was measured in the reaction mixture adjusted to pH 5.5–8.5 as described under “Experimental Section.” (C,D) Temperature dependence and temperature stability of recFlRed. These measurements were also performed as described in the “Experimental Section.”
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-00493-f003: Effects of pH and temperature on the activity of recFlRed. (A) SDS-polyacrylamide gel electrophoresis for recFlRed. Mk, marker proteins; (B) pH dependence of recFlRed. Activity was measured in the reaction mixture adjusted to pH 5.5–8.5 as described under “Experimental Section.” (C,D) Temperature dependence and temperature stability of recFlRed. These measurements were also performed as described in the “Experimental Section.”

Mentions: To examine if the translation product of flred exhibits the DEH-reductase activity, we produced recFlRed with an Escherichia coli expression system (see Experimental Section 4.3). As shown in Figure 3A, apparent molecular mass of recFlRed was estimated to be ~28 kDa by SDS-PAGE, which was consistent with the molecular mass calculated from the amino-acid sequence. The yield of recFlRed was considerably high, i.e., ~40 mg of recFlRed could be produced in 1 L of E. coli culture. To determine co-factor specificity of recFlRed, the DEH-reducing activity was examined in the presence of either NADH or NADPH. As shown in Table 1, specific activities of recFlRed in the presence of NADH and NADPH were found to be 4.0 U/mg and 0.043 U/mg, respectively. This indicated that FlRed was a NADH-specific enzyme as predicted by the amino-acid sequence analysis. Optimal pH and temperature of recFlRed were observed at around 7.2 and 25 °C (Figure 3B,C), respectively. Thus, recFlRed was not so heat-stable, e.g., the activity decreased to half of the original level by the incubation at ~28 °C for 30 min (Figure 3D). Then, substrate specificity of recFlRed was examined with a variety of substrates such as aldehyde, ketone, keto ester, α-keto acid and aldose (Table 1). As a result, recFlRed was found to show appreciably no activity to such substrates. Accordingly, we concluded that recFlRed was the DEH reductase with high specificity to NADH.


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)

Effects of pH and temperature on the activity of recFlRed. (A) SDS-polyacrylamide gel electrophoresis for recFlRed. Mk, marker proteins; (B) pH dependence of recFlRed. Activity was measured in the reaction mixture adjusted to pH 5.5–8.5 as described under “Experimental Section.” (C,D) Temperature dependence and temperature stability of recFlRed. These measurements were also performed as described in the “Experimental Section.”
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-00493-f003: Effects of pH and temperature on the activity of recFlRed. (A) SDS-polyacrylamide gel electrophoresis for recFlRed. Mk, marker proteins; (B) pH dependence of recFlRed. Activity was measured in the reaction mixture adjusted to pH 5.5–8.5 as described under “Experimental Section.” (C,D) Temperature dependence and temperature stability of recFlRed. These measurements were also performed as described in the “Experimental Section.”
Mentions: To examine if the translation product of flred exhibits the DEH-reductase activity, we produced recFlRed with an Escherichia coli expression system (see Experimental Section 4.3). As shown in Figure 3A, apparent molecular mass of recFlRed was estimated to be ~28 kDa by SDS-PAGE, which was consistent with the molecular mass calculated from the amino-acid sequence. The yield of recFlRed was considerably high, i.e., ~40 mg of recFlRed could be produced in 1 L of E. coli culture. To determine co-factor specificity of recFlRed, the DEH-reducing activity was examined in the presence of either NADH or NADPH. As shown in Table 1, specific activities of recFlRed in the presence of NADH and NADPH were found to be 4.0 U/mg and 0.043 U/mg, respectively. This indicated that FlRed was a NADH-specific enzyme as predicted by the amino-acid sequence analysis. Optimal pH and temperature of recFlRed were observed at around 7.2 and 25 °C (Figure 3B,C), respectively. Thus, recFlRed was not so heat-stable, e.g., the activity decreased to half of the original level by the incubation at ~28 °C for 30 min (Figure 3D). Then, substrate specificity of recFlRed was examined with a variety of substrates such as aldehyde, ketone, keto ester, α-keto acid and aldose (Table 1). As a result, recFlRed was found to show appreciably no activity to such substrates. Accordingly, we concluded that recFlRed was the DEH reductase with high specificity to NADH.

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