The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-Å resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main-and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related ␤-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.Homoisocitrate dehydrogenase (HICDH) is the third enzyme involved in lysine biosynthesis through ␣-aminoadipate (19) and is a member of a family of ␤-decarboxylating dehydrogenases that includes isocitrate dehydrogenase (ICDH) in the tricarboxylic acid cycle, 3-isopropylmalate dehydrogenase (IPMDH) in leucine biosynthesis, and tartrate dehydrogenase in vitamin production (2). It has been suggested that these enzymes diverged from a common ancestral ␤-decarboxylating dehydrogenase (4, 9, 34).We have shown that in an extremely thermophilic bacterium, Thermus thermophilus HB27, lysine is synthesized through ␣-aminoadipate, although lysine is synthesized through diaminopimelate in most bacteria (13,18,20,22,23,31). We have previously characterized HICDH from T. thermophilus HB27 (TtHICDH) in detail and have shown that the enzyme has four unique features (19). The first is its substrate specificity. Although TtHICDH is essential for lysine biosynthesis in the bacterium, TtHICDH can catalyze the reaction with isocitrate as a substrate; isocitrate is the substrate of ICDH at a 20-fold-higher efficiency than toward the native substrate, homoisocitrate. This feature contrasts with HICDH from Saccharomyces cerevisiae, which utilizes only homoisocitrate as a substrate (19). Moreover, the substrate specificity is easily altered by a single mutation, Arg85Val, to lose activity for isocitrate but to acquire activity for 3-isopropylmalate, a substrate of IPMDH. The second feature is the unique subunit organization. TtHICDH is a homotetramer, while mo...

This study evaluated the effect of 13 years of swine‐manure application on the changes in soil hydraulic properties, and as associated physicochemical properties, with a focus on heavy metal mobility. Various soil hydraulic properties were measured, including soil water retention (SWR), saturated field hydraulic conductivity (Kfs) and unsaturated field hydraulic conductivity (Kfunsat) using a disc infiltrometer. Heavy metal mobility was evaluated with a sequential extraction procedure. At 0–30 cm soil depth in the heavily manured plot (SMhigh plot), SWR at 0 to −100 kPa was significantly larger than in plots amended with a standard amount of manure (SMstd plot) or with chemical fertilizer (CF plot). Kfs and Kfunsat values in both manure‐amended plots were less than in the CF plot under dry soil conditions but greater than those of the CF plot under wet soil conditions. Furthermore, Kfs and Kfunsat did not necessarily increase with manure application rates. On the other hand, high‐mobility metal fractions, such as the exchangeable fraction of Zn, and the CH3CO2Na‐extractable fraction of Zn and Mn, and the metal–organic complex fractions of Zn, Cu and Mn, increased with the greater manure application rate. In addition, low‐mobility metal fractions, the organically bound fractions of Zn, Cu and Mn in the high SM plot and the easily reducible metal oxide fraction of Mn in both manure‐amended plots were probably affected and released into high‐mobility fractions. This indicated that manure application changed the soil redox conditions by improving the soil structure, depending on the water content of soil pores. Despite the reduction of Kfs and Kfunsat by heavy manure application, the transport of high‐mobility metal fractions with either surface water flow or infiltration water flow could be controlled by soil water content at the beginning of a rain or irrigation event.

Applying manure compost not only results in zinc accumulation in the soil but also causes an increase in zinc mobility and enhances zinc leaching. In this study, the physical and chemical characteristics of zinc, zinc profiles, and zinc balance were investigated to characterise the fate of zinc in fields where the quality and amount of pig manure compost applied have been known for 13 years. Moreover, we determined zinc fractionation in both 0.1 mol L(-1)HCl-soluble (mobile) and -insoluble (immobile) fractions. Adsorption of zinc in the soil was enhanced with increasing total carbon content following the application of pig manure compost. The 159.6 mg ha(-1) year(-1)manure applied plot (triplicate) exceeded the Japanese regulatory level after only 6 years of applying pig manure compost, whereas the 53.2 mg ha(-1) year(-1) manure applied plot (standard) reached the regulatory level after 13 years. The zinc loads in the plots were 17.0 and 5.6 kg ha(-1) year(-1), respectively. However, 5.9 % and 17.2 % of the zinc loaded in the standard and the triplicate pig manure compost applied plots, respectively, were estimated to be lost from the plough layer. Based on the vertical distribution of mobile and immobile zinc content, a higher rate of applied manure compost caused an increase in the mobile zinc fraction to a depth of 40 cm. Although the adsorption capacity of zinc was enhanced following the application of pig manure compost, a greater amount of mobile zinc could move downward through the manure amended soil than through non manure-amended soil.

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HICDH (homoisocitrate dehydrogenase), which is involved in lysine biosynthesis through α-aminoadipate, is a paralogue of IPMDH [3-IPM (3-isopropylmalate) dehydrogenase], which is involved in leucine biosynthesis. TtHICDH (Thermus thermophilus HICDH) can recognize isocitrate, as well as homoisocitrate, as the substrate, and also shows IPMDH activity, although at a considerably decreased rate. In the present study, the promiscuous TtHICDH was evolved into an enzyme showing distinct IPMDH activity by directed evolution using a DNA-shuffling technique. Through five repeats of DNA shuffling/screening, variants that allowed Escherichia coli C600 (leuB⁻) to grow on a minimal medium in 2 days were obtained. One of the variants LR5-1, with eight amino acid replacements, was found to possess a 65-fold increased k(cat)/K(m) value for 3-IPM, compared with TtHICDH. Introduction of a single back-replacement H15Y change caused a further increase in the k(cat)/K(m) value and a partial recovery of the decreased thermotolerance of LR5-1. Site-directed mutagenesis revealed that most of the amino acid replacements found in LR5-1 effectively increased IPMDH activity; replacements around the substrate-binding site contributed to the improved recognition for 3-IPM, and other replacements at sites away from the substrate-binding site enhanced the turnover number for the IPMDH reaction. The crystal structure of LR5-1 was determined at 2.4 Å resolution and revealed that helix α4 was displaced in a manner suitable for recognition of the hydrophobic γ-moiety of 3-IPM. On the basis of the crystal structure, possible reasons for enhancement of the turnover number are discussed.