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A novel enzyme catalysing citryl-CoA cleavage to acetyl-CoA and oxaloacetate was purified from Hydrogenobacter thermophilus TK-6, and designated citryl-CoA lyase (CCL). The citrate cleavage reaction in this organism proceeded by a unique set of two consecutive reactions: (i). citryl-CoA formation by citryl-CoA synthetase (CCS) and (ii). citryl-CoA cleavage by CCL. Purified CCL gave a single 30 kDa band in SDS-PAGE and gel filtration chromatography indicated that the native state of the enzyme exists as a trimer (alpha(3)). Citryl-CoA lyase showed low citrate synthase (CS) activity. Using an oligonucleotide probe, the corresponding gene was cloned and sequenced. The gene was expressed in Escherichia coli and recombinant CCL was also purified. The CCL protein sequence showed similarity to the C-terminal regions of ATP citrate lyase (ACL) and CS sequences in the database. By further sequence comparisons, the phylogenetic relationship between CCS, CCL, ACL and CS was investigated.

Male Sprague-Dawley rats, 4 weeks of age, were fed purified diets either with or without 0.2% soy isoflavones rich powder for 5 weeks to elucidate their direct functions such as antioxidative action and regulation of lipid metabolism. Dietary soy isoflavones decreased serum lipid peroxide level in rats. Levels of liver and serum alpha-tocopherol were higher in the rats fed isoflavone than in those fed isoflavones-free diet. Thus, dietary soy isoflavones exhibited mild antioxidative function in this animal experiment. Isoflavone metabolites from diet may act as scavengers of reactive oxygen species. Dietary soy isoflavones lowered hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity, although liver cholesterol level was not modulated. However, the levels of serum cholesterol and triglyceride decreased by consumption of soy isoflavones. Therefore, dietary soy isoflavones may exhibit hypocholesterolemic and hypolipidemic functions. Moreover, dietary soy isoflavones lowered hepatic Delta6 desaturase activity. Reflecting this observation, Delta6 desaturation indices ((18:2(n = 6) + 18:3(n = 6))/20:4(n = 6)) of tissue lipids tended to be lower in rats fed isoflavones than in those fed isoflavones-free diet. This action may contribute to the prevention of inflammatory response by imbalance of eicosanoids. These observations suggest that the positive intake of soy isoflavones may reduce the risk of some cardiovasucular diseases through their radical scavenging function and hypocholesterolemic action.

Isocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters. Notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2-oxoglutarate. The carboxylation reaction required the addition of cell extract and ATP-Mg, suggesting the existence of additional carboxylation factor(s). Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides. N-terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation. Pyruvate carboxylase was also purified, but was not active in stimulating isocitrate dehydrogenase. Isocitrate dehydrogenase, the novel biotin protein, ATP-Mg and NADH were essential for the reductive carboxylation of 2-oxoglutarate. These observations indicate that the novel biotin protein is an ATP-dependent factor, which is involved in the reverse (carboxylating) reaction of isocitrate dehydrogenase.

A 7.8 kb fragment containing isocitrate dehydrogenase (ICDH) gene and its flanking regions was cloned from Escherichia coli BL21(DE3) and sequenced. Unlike the case of the K-12 strain, the e14 element was not found. The nucleotide divergence between these two strains was about 2%. Using the cloned fragment, ICDH defective mutant strain, MA1935, was generated from BL21(DE3). Although MA1935 accumulated citrate, citrate synthase activity was not repressed but was rather high. In addition, isocitrate lyase was not highly induced at the stationary phase. MA1935 was shown to be a good host strain for ICDH gene expression., (Copyright 2003 Wiley Periodicals Inc.)

Pseudomonas aeruginosa strains deficient in the genes for cytochrome c1, a subunit of the cytochrome bc1 complex, or the tetraheme membrane protein NapC, which is similar to NirT of Pseudomonas stutzeri, were constructed and their growth was investigated. The cytochrome c1 mutant could not grow under anaerobic conditions with nitrite as an electron acceptor and did not reduce nitrite in spite of its producing active nitrite reductase. NirM (cytochrome c551) and azurin, which are the direct electron donors for nitrite reductase, were reduced by succinate in the presence of the membrane fraction from the wild-type strain as a mediator but not in the presence of that from the cytochrome c1 mutant. These results indicated that cytochrome bc1 complex was necessary for electron transfer from the membrane quinone pool to nitrite reductase. The NapC mutant grew anaerobically at the expense of nitrite, indicating that NapC was not necessary for nitrite reduction.

Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6. The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains. Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions. The obtained differences in the Gibbs free energy changes of unfolding [Delta(DeltaG)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change (DeltaC(P)), resulting in higher DeltaG values over a wide temperature range (0-100 degrees C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552. Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization.

Four groups of microbial mix culture with considerable capability of cellulose degradation were selected from four compost heaps, by combining the four groups, a composite microbial system MC1 with highly efficient cellulose degradation was obtained. It was found that 0.48 g of filter paper, 0.49 g of absorbent cotton, 0.19 g of wheat straw, or 0.08 g of wood residue can be degraded by 100 mL of MC1 at 50 degrees C with in 72 hours under static culture. The CM1 saccharification activity was much higher at 24th hours when degrading filter paper that was 122.3 U.mL-1. This capability of degradation maintained more than 20 days when the substrates were continually added. MC1 could be inoculated in a wide pH rang, from 4 to 10; however, the final pH would be changed to neutrality after incubation. The pH would be stability with filter paper between 6.0 and 6.5, and between 8.0 and 8.5 without the filter paper.

CbbQ is encoded by the gene located downstream of ribulose 1,5-bisphosphate carboxylase/oxygenase genes (cbbLS) in the thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus. The protein possesses two nucleotide-binding motifs in its amino acid sequence, and it posttranslationally activates RubisCO. We present ATP hydrolysis and binding of CbbQ. CbbQ releases P(i) from ATP only in the presence of Mg(2+). CbbQ interacts with an 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate in the presence or absence of Mg(2+). The interaction with Mg(2+) and/or a nucleotide induces a conformational change in CbbQ. Autophosphorylation of CbbQ occurs only in the absence of Mg(2+)., (©2002 Elsevier Science (USA).)