Identification of a 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid Reductase, FlRed, in an Alginolytic Bacterium Flavobacterium sp. Strain UMI-01

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.

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate

“…Essentially the same results have been obtained in the reduction of DEH to KDG by FlRed (23). To confirm the formation of KDG by HdRed, chemical modification analysis with DMB (35) was performed.…”

“…DEH was prepared by the digestion of sodium alginate with crude enzyme from Flavobacterium sp. UMI-01 as reported previously (23). TOYOPEARL Butyl-650M, TOYOPEARL DEAE-650M, and TOYOPEARL QAE-550C were purchased from Toyo Soda Mfg.…”

“…HdRed-NdeF, 5Ј-GAAGGAGATATACATATGGCGGCGGTACCAGAAGA-3Ј; HdRed-inf8G8H-R, 5Ј-CACCTCCACCGGATCCCCCGG-AACTGTTCTGTACGG-3Ј, respectively) with PrimeStarMax DNA polymerase (Takara). Amplified DNA was subcloned into the NdeI-XbaI cloning site of pT7-7 plasmid, which had been modified for adding the His 8 tag to the C terminus of recombinant proteins as in our previous report (23). The pT7-7-HdRed recombinant plasmid was transfected to E. coli BL21(DE3) and cultured at 37°C for 16 h in 2 liters of 2ϫ YT medium and further incubated at 20°C for 1 h. Then expression of recombinant HdRed (rHdRed) was induced by the addition of isopropyl 1-thio-␤-D-galactopyranoside to make the final concentration 0.1 mM.…”

“…1) and then metabolized to glyceraldehyde-3-phosphate and pyruvate via the Entner-Doudoroff pathway (19,20). Recently, DEH-specific reductases, A1-R (21), A1-RЈ (22), and FlRed (23), were identified in alginolytic bacteria Sphingomonas sp. strain A1 and Flavobacterium sp.…”

Continuous Saccharification of Laminarin by Immobilized Laminarinase ULam111 Followed by Ethanol Fermentation with a Marine-Derived Yeast

“…The thermal cycling conditions were: initial denaturation at 98˚C for 30 sec, 30 cycles of denaturation at 98˚C for 10 sec, annealing at 55˚C for 15 sec, and extension at 72˚C for 45 sec. Amplified DNA was ligated into a modified pCold vector (Takara, Shiga, Japan), which has been reported [23], using the In-Fusion HD Cloning Kit (Takara). After a sequencing analysis, recombinant plasmid DNA was introduced to E. coli BL21 (DE3) (Nippon Gene) or E. coli Rosetta-gami™ 2 cells (Novagen, Darmstadt, Germany).…”