The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the Cterminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2⅐E3BP was thought to be 60 E2 plus ϳ12 E3BP. We have prepared homogenous human components. E2 and E2⅐E3BP have s 20,w values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2⅐E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2⅐E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound ϳ60 E1 and maximally sedimented 64.4 ؎ 1.5 S faster than E2, whereas E1-saturated E2⅐E3BP maximally sedimented 49.5 ؎ 1.4 S faster than E2⅐E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (ϳ12) were bound by E2⅐E3BP than by E2. The findings of a smaller E2⅐E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E2 48 ⅐E3BP 12 mass, which is consistent with this model.The mitochondrial pyruvate dehydrogenase complex (PDC) 1 catalyzes the irreversible conversion of pyruvate to acetyl-CoA along with the reduction of NAD ϩ . PDCs from all known sources contain the pyruvate dehydrogenase (E1), the dihydrolipoyl acetyltransferase (E2), and the dihydrolipoyl dehydrogenase (E3) components. Mammalian PDC has a highly organized structure in which the E2 component plays a central role in the organization, integrated chemical reactions, and regulation of the complex (1-5). Besides those universal components, the mammalian and subsequently some other eukaryotic PDCs were shown to contain another component, called E3-binding protein (E3BP); this protein was originally designated protein X (6, 7). Mammalian E3BP was first characterized as a component with a reactive lipoyl group on a single lipoyl domain (7-11) that, alone, could support the overall reaction (12). E3BP was tightly bound to E2 (7) by its C-terminal region (10). The E3BP component was then shown to contribute to the organization of the complex by binding the E3 component (13)(14)(15)(16)(17)(18). This work provides new insights into the integration of E3BP into the central framework of the mammalian complex.Although first characterized in the bo...

Staphylococcus warneri ISK-1 produces a lantibiotic, nukacin ISK-1. The nukacin ISK-1 gene cluster consists of at least six genes, nukA, -M, -T, -F, -E, and -G, and two open reading frames, ORF1 and ORF7 (designated nukH). Sequence comparisons suggested that NukF, -E, -G, and -H contribute to immunity to nukacin ISK-1. We investigated the immunity levels of recombinant Lactococcus lactis expressing nukFEG and nukH against nukacin ISK-1. The co-expression of nukFEG and nukH resulted in a high degree of immunity. The expression of either nukFEG or nukH conferred partial immunity against nukacin ISK-1. These results suggest that NukH contributes cooperatively to self-protection with Nuk-FEG. The nukacin ISK-1 immunity system might function against another lantibiotic, lacticin 481. Western blot analysis showed that NukH expressed in Staphylococcus carnosus was localized in the membrane. Peptide release/bind assays indicated that the recombinant L. lactis expressing nukH interacted with nukacin ISK-1 and lacticin 481 but not with nisin A. These findings suggest that NukH contributes cooperatively to host immunity as a novel type of lantibiotic-binding immunity protein with NukFEG.

Small heat shock protein (sHSP) is a family of ubiquitous polypeptides involved in a variety of physiological phenomena. From the silkworm, Bombyx mori, we isolated and sequenced the following cDNAs encoding sHSPs: shsp19.9, shsp20.1, shsp20.4, shsp20.8, shsp21.4, and shsp23.7. shsp21.4 was nearly twice as large in size as other shsps. The deduced amino acid sequence of sHSP21.4 was similar to that of Drosophila melanogaster CG14207-PA. Other sHSPs were highly similar to each other and, in a phylogenetic tree, formed a cluster including Plodia interpunctella alphaCP25. It was speculated that shsp21.4 has at least one intron in genome while other shsps do not. The transcripts of all shsps were subtle, but were constitutively detected in various tissues. Heat shock triggered a substantial increase in the transcripts other than shsp21.4. The B. mori sHSPs are perhaps classified into at least two groups: sHSP21.4 and others.

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.

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A cDNA encoding glutathione S-transferase (GST) of the fall webworm, Hyphantria cunea, was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone (hcGST) was sequenced and deduced for amino acid sequence, which revealed 87, 59, and 42% identities to Sigma-class GSTs from Bombyx mori, Manduca sexta, and Blattella germanica respectively. A recombinant hcGST protein (rhcGST) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rhcGST retained more than 75% of its original GST activity after incubation at pHs 6 to 11. Incubation for 30 min at temperatures below 50 degrees C scarcely affected the activity. rhcGST was able to catalyze the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. We also found that as compared to B. mori Sigma-class GST, rhcGST had a higher affinity for fenitrothion, an organophosphorus insecticide.

An Escherichia coli system was engineered for the heterologous production of itaconic acid via the expression of cis-aconitate decarboxylase gene (cad), and then maximal itaconic acid levels produced by engineered E. coli were evaluated. Expression of cad in E. coli grown in Luria-Bertani (LB) medium without glucose in a test tube resulted in 0.07 g/L itaconic acid production after 78 h at 20 C. To increase itaconic acid production, E. coli recombinants were constructed by inactivating the isocitrate dehydrogenase gene (icd) and/or the isocitrate lyase gene (aceA). Expression of cad and inactivation of icd resulted in 0.35 g/L itaconic acid production after 78 h, whereas aceA inactivation had no effect on itaconic acid production. The intracellular itaconate concentration in the icd strain was higher than that in the cad-expressing strain without icd inactivation, which suggests that the extracellular secretion of itaconate in E. coli is the rate-determining step during itaconic acid production. pH-stat cultivation using the cad-expressing icd strain in LB medium with 3% glucose in a jar fermenter resulted in 1.71 g/L itaconic acid production after 97 h at 28 C. To further increase itaconic acid production, the aconitase B gene (acnB) was overexpressed in the cad-expressing icd strain. Simultaneous overexpression of acnB with the expression of cad in the icd strain led to 4.34 g/L itaconic acid production after 105 h. Our findings indicate that icd inactivation and acnB overexpression considerably enhance itaconic acid production in cad-expressing E. coli.Key words: aconitase; cis-aconitate decarboxylase; isocitrate dehydrogenase; isocitrate lyase; itaconic acid; metabolic engineering IntroductionOver the past several decades, there has been substantial interest in itaconic acid as a dicarboxyvinyl monomer produced by microbial fermentation of biomass (Kobayashi and Nakamura, 1964;Okabe et al., 2009;Willke and Vorlop, 2001). Owing to its useful properties, itaconic acid is used in the manufacturing of synthetic polymers, such as plastics and resins (Milson and Meers, 1985;Tate, 1981). Graft polymers with an itaconic acid-based main chain and oligolactate side chain have been synthesized and characterized in our laboratory Okada et al., 2012). The polymers consisting of itaconic acid had high biomass content and are therefore thought to contribute to carbon emission reduction.Itaconic acid is industrially produced from sugars, such as glucose, by the fungus Aspergillus terreus via submerged fermentation (Bonnarme et al., 1995). To date, many re searchers have reported increased productivity of a native itaconic acid producer, A. terreus, by using techniques associated with mutation breeding (Yahiro et al., 1995) and fermentation conditions (Okabe et al., 1993;Park et al., 1994;Riscaldati et al., 2000;Träger et al., 1989;Yahiro et al., 1997). Specifically, the spontaneous mutant A. terreus TN-484 is used as a target microorganism for optimizing the fermentation process. In a previous study, a typical yield of 82....

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