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-erythro-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.

Highlights

  • Alginate is a viscous and gel-forming polysaccharide comprising β-D-mannuronic acid (M) and α-L-guluronic acid (G), which form poly (M), poly (G) and random (MG) blocks in the alginate polymer [1,2,3,4]

  • 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]; in this bacterium, it may participate in alginate metabolism

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Summary

Introduction

Alginate is a viscous and gel-forming polysaccharide comprising β-D-mannuronic acid (M) and α-L-guluronic acid (G), which form poly (M), poly (G) and random (MG) blocks in the alginate polymer [1,2,3,4]. An end product of alginate lyases (EC 4.2.2.3, EC 4.2.2.11), i.e., 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH), was recently used as a carbon source for ethanol fermentation with genetically modified microorganisms [13,14,15]. This implies that alginate may become available as a biomass for bioethanol production along with molasses, starch and cellulosic biomasses. To realize the production of bioethanol from alginate, efficient fermentation systems for this polysaccharide should be established through extensive understanding of its metabolic pathway

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