Kousaku Murata

近年，循環型社会の構築のため，海洋バイオマスの利活用が重要な課題の一つとなっている．特に，褐藻類の主要な成分であるヘテロ多糖アルギン酸は有望なバイオマスとして期待されている．そのため，アルギン酸資化性細菌を中心に，その分解酵素の研究が盛んに行われているが，細胞内取り込み系はよくわかっていない．菌体外に分解酵素を分泌する多くのアルギン酸資化性細菌とは異なり，Sphingomonas属細菌A1株はアルギン酸を「超分子」を介して高分子のまま取り込み資化する．最近，「超分子」の主要な構成要素であるABCトランスポーターの立体構造が決定され，その構造的特徴から高分子の輸送を可能にする仕組みがわかってきた．本稿では，多糖アルギン酸の取り込みに機能する結合タンパク質と輸送体およびアルギン酸代謝酵素を中心に，それらの構造基盤について紹介する．

NAD kinase (NADK) is a crucial enzyme for production of NADP+. ATP-specific NADK prefers ATP to inorganic polyphosphate [lsqb]poly(P)[rsqb] as a phosphoryl donor, whereas poly(P)/ATP-NADK utilizes both ATP and poly(P), and is employed in industrial mass production of NADP+. Poly(P)/ATP-NADKs are distributed throughout Gram-positive bacteria and Archaea, whereas ATP-specific NADKs are found in Gram-negative [agr]- and [ggr]-proteobacteria and eukaryotes. In this study, we succeeded in conferring the ability to utilize poly(P) on [ggr]-proteobacterial ATP-specific NADKs through a single amino-acid substitution; the substituted amino-acid residue is therefore important in determining the phosphoryl-donor specificity of [ggr]-proteobacterial NADKs. We also demonstrate that a poly(P)/ATP-NADK created through this method is suitable for the poly(P)-dependent mass production of NADP+. Moreover, based on our results, we provide insight into the evolution of bacterial NADKs, in particular, how NADKs evolved from poly(P)/ATP-NADKs into ATP-specific NADKs.

Bioethanol has attracted attention as an alternative to petroleum-derived fuel. Seaweeds have been proposed as some of the most promising raw materials for bioethanol production because they have several advantages over lignocellulosic biomass. However, because seaweeds contain low contents of glucans, i.e., polysaccharides composed of glucose, the conversion of only the glucans from seaweed is not sufficient to produce high concentrations of ethanol. Therefore, it is also necessary to produce ethanol from other specific carbohydrate components of seaweeds, including sulfated polysaccharides, mannitol, alginate, agar and carrageenan. This review summarizes the current state of research on the production of ethanol from seaweed carbohydrates for which the conversion of carbohydrates to sugars is a key step and makes comparisons with the production of ethanol from lignocellulosic biomass. This review provides valuable information necessary for the production of high concentrations of ethanol from seaweeds.

NAD ⁺ is synthesized from tryptophan either via the kynurenine ( de novo ) pathway or via the salvage pathway by reutilizing intermediates such as nicotinic acid or nicotinamide ribose. Quinolinic acid is an intermediate in the kynurenine pathway. We have discovered that the budding yeast Saccharomyces cerevisiae secretes quinolinic acid into the medium and also utilizes extracellular quinolinic acid as a novel NAD ⁺ precursor. We provide evidence that extracellular quinolinic acid enters the cell via Tna1, a high-affinity nicotinic acid permease, and thereby helps to increase the intracellular concentration of NAD ⁺ . Transcription of genes involved in the kynurenine pathway and Tna1 was increased, responding to a low intracellular NAD ⁺ concentration, in cells bearing mutations of these genes; this transcriptional induction was suppressed by supplementation with quinolinic acid or nicotinic acid. Our data thus shed new light on the significance of quinolinic acid, which had previously been recognized only as an intermediate in the kynurenine pathway.

Alginate is a heteropolysaccharide that consists of β-d-mannuronate (M) and α-l-guluronate (G). The Gram-negative bacterium Sphingomonas sp. A1 directly incorporates alginate into the cytoplasm through the periplasmic solute-binding protein (AlgQ1 and AlgQ2)-dependent ABC transporter (AlgM1-AlgM2/AlgS-AlgS). Two binding proteins with at least four subsites strongly recognize the nonreducing terminal residue of alginate at subsite 1. Here, we show the broad substrate preference of strain A1 solute-binding proteins for M and G present in alginate and demonstrate the structural determinants in binding proteins for heteropolysaccharide recognition through X-ray crystallography of four AlgQ1 structures in complex with saturated and unsaturated alginate oligosaccharides. Alginates with different M/G ratios were assimilated by strain A1 cells and bound to AlgQ1 and AlgQ2. Crystal structures of oligosaccharide-bound forms revealed that in addition to interaction between AlgQ1 and unsaturated oligosaccharides, the binding protein binds through hydrogen bonds to the C4 hydroxyl group of the saturated nonreducing terminal residue at subsite 1. The M residue of saturated oligosaccharides is predominantly accommodated at subsite 1 because of the strict binding of Ser-273 to the carboxyl group of the residue. In unsaturated trisaccharide (ΔGGG or ΔMMM)-bound AlgQ1, the protein interacts appropriately with substrate hydroxyl groups at subsites 2 and 3 to accommodate M or G, while substrate carboxyl groups are strictly recognized by the specific residues Tyr-129 at subsite 2 and Lys-22 at subsite 3. Because of this substrate recognition mechanism, strain A1 solute-binding proteins can bind heteropolysaccharide alginate with different M/G ratios.

Sphingomonas sp. A1 directly incorporates alginate polysaccharides through a `superchannel' comprising a pit on the cell surface, alginate-binding proteins in the periplasm and an ABC transporter (alginate importer) in the inner membrane. Alginate importer, consisting of four subunits, AlgM1, AlgM2 and two molecules of AlgS, was crystallized in the presence of the binding protein AlgQ2. Preliminary X-ray analysis showed that the crystal diffracted to 3.3 Å resolution and belonged to space group P2₁2₁2₁, with unit-cell parameters a = 72.5, b = 136.8, c = 273.3 Å, suggesting the presence of one complex in the asymmetric unit.