Tartrate dehydrogenase reductive decarboxylation: stereochemical generation of diastereotopically deuterated hydroxymethylenes

Tartrate dehydrogenase catalyzes the reductive decarboxylation of meso-tartrate to glycerate. Concomitant with the ketonization of the intermediate enolate the C3 hydroxymethylene of glycerate necessarily acquires a proton from solvent. In D2O, the proton is shown to be added stereospecifically to form (2R,3R)-[3-2H]glycerate. The 1H-NMR assignments of the diastereotopic C3 protons of glycerate were confirmed by the enzymatic conversion of [1R-2H]fructose-6-phosphate to (2R,3R)-[3-2H]glycerate. The decarboxylation-protonation occurs with retention of configuration, implying that the general acid is positioned on the same face of the intermediate as the departing carboxylate. The stereochemically pure (2R,3R)-[3-2H]glycerate is readily synthesized and serves as a chiral hydroxymethylene synthon as demonstrated by the synthesis of (2S,3R)-[3-2H]serine.     

Structural diversity and functional novelty of new carotenoid biosynthesis genes

Many new carotenoid synthesis genes have recently been identified through genomic sequencing or functional cloning. Some of them exhibit novel structures and/or novel functions. This review describes such examples in the families of lycopene β-cyclases, putative homologues of phytoene dehydrogenases and new carotenoid hydroxylases. Both the functionally novel lycopene β-monocyclases and structurally novel fusion-type of lycopene β-cyclases were described. Another newly discovered sequence of lycopene β-cyclase described might represent a new class of lycopene β-cyclases previously not identified in several cyanobacteria. Three examples of putative homologues of phytoene dehydrogenases were described, however, they were confirmed to encode different and/or new functions such as β-carotene ketolase, 4,4′-diapolycopene oxygenase or prolycopene isomerase. Two new carotenoid hydroxylase genes were described that encoded the new function of 2,2′-β-ionone ring hydroxylase or 3,3′-isorenieratene hydroxylase. Phylogenetic analysis of these genes shed light on their possible evolutionary origins. These new genes also provide tools for synthesis of novel and desirable carotenoids by genetic engineering.     

Co-expression of P450 BM3 and glucose dehydrogenase by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in an NADPH-dependent indigo production system

P450 BM3 mutant can catalyze indole to indoxyl, and indoxyl can dimerize to form indigo. But the reaction catalyzed by P450 BM3 requires NADPH, as coenzyme regeneration is very important in this system. As we know, when glucose dehydrogenase oxidizes glucose to glucolactone, NADH or NADPH can be formed, which can contribute to NADPH regeneration in the reaction catalyzed by P450 BM3. In this paper, a recombinant Escherichia coli BL21 (pET28a (+)-P450 BM3-gdh0310) was constructed to co-express both P450 BM3 gene and glucose dehydrogenase (GDH) gene. To improve the expression level of P450 BM3 and GDH in E. coli and to avoid the complex and low-efficiency refolding operation in the purification procedure, the expression conditions were optimized. Under the optimized conditions, the maximum P450 BM3 and GDH activities amounted to 8173.13 and 0.045 U/mg protein, respectively. Then bioconversion of indole to indigo was carried out by adding indole and glucose to the culture after improved expression level was obtained under optimized conditions, and 2.9 mM (760.6 mg/L) indigo was formed with an initial indole concentration of 5 mM.     

Distinct modes of recognition of the lipoyl domain as substrate by the E1 and E3 components of the pyruvate dehydrogenase multienzyme complex

Two-dimensional (15)N-heteronuclear single-quantum coherence (HSQC) NMR studies with a di-domain (lipoyl domain+ linker+ peripheral subunit-binding domain) of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus allowed a molecular comparison of the need for lipoic acid to be covalently attached to the lipoyl domain in order to undergo reductive acetylation by the pyruvate decarboxylase (E1) component, in contrast with the ability of free lipoic acid to serve as substrate for the dihydrolipoyl dehydrogenase (E3) component. Tethering the lipoyl domain to the peripheral subunit-binding domain in a complex with E1 or E3 rendered the system more like the native enzyme complex, compared with the use of a free lipoyl domain, yet of a size still amenable to investigation by NMR spectroscopy. Recognition of the tethered lipoyl domain by E1 was found to be ensured by intensive interaction with the lipoyl-lysine-containing beta-turn and with residues in the protruding loop close to the beta-turn. The size and sequence of this loop varies significantly between species and dictates the lipoylated lipoyl domain as the true substrate for E1. In contrast, with E3 the main interaction sites on the tethered lipoyl domain were revealed as residues Asp41 and Ala43, which form a conserved sequence motif, DKA, around the lipoyl-lysine residue. No domain specificity is observed at this step and substrate channelling in the complex thus rests on the recognition of the lipoyl domain by the first enzyme, E1. The cofactor, thiamine diphosphate, and substrate, pyruvate, had distinct but contrasting effects on the E1/di-domain interaction, whereas NAD(+) and NADH had negligible effect on the E3/di-domain interaction. Tethering the lipoyl domain did not significantly change the nature of its interaction with E1 compared with a free lipoyl domain, indicative of the conformational freedom allowed by the linker in the movement of the lipoyl domain between active sites.     

Berberine alters the processing of Alzheimer's amyloid precursor protein to decrease Abeta secretion

Berberine is an isoquinoline alkaloid isolated from Coptidis rhizoma, a major herb widely used in Chinese herbal medicine. Berberine's biological activity includes antidiarrheal, antimicrobial, and anti-inflammatory effects. Recent findings show that berberine prevents neuronal damage due to ischemia or oxidative stress and that it might act as a novel cholesterol-lowering compound. The accumulation of amyloid-beta peptide (Abeta) derived from amyloid precursor protein (APP) is a triggering event leading to the pathological cascade of Alzheimer's disease (AD); therefore the inhibition of Abeta production should be a rational therapeutic strategy in the prevention and treatment of AD. Here, we report that berberine reduces Abeta levels by modulating APP processing in human neuroglioma H4 cells stably expressing Swedish-type of APP at the range of berberine concentration without cellular toxicity. Our results indicate that berberine would be a promising candidate for the treatment of AD.     

A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors

ATP-gated P2X4 receptors (P2X4R) are abundantly expressed in the CNS. However, little is known about the molecular targets for ethanol action in P2X4Rs. The current investigation tested the hypothesis that the ectodomain-transmembrane (TM) interface contains residues that are important for the action of ethanol in P2X4Rs. Wild type (WT) and mutant P2X4R were expressed in Xenopus oocytes. ATP concentration–response curves and ethanol (10–200 mM)-induced changes in ATP EC₁₀-gated currents were determined using two-electrode voltage clamp (−70 mV). Alanine substitution at the ectodomain-TM1 interface (positions 50–61) resulted in minimal changes in ethanol response. On the other hand, alanine substitution at the ectodomain-TM2 interface (positions 321–337) identified two key residues (D331 and M336) that significantly reduced ethanol inhibition of ATP-gated currents without causing marked changes in ATP I _max, EC₅₀, or Hill's slope. Other amino acid substitutions at positions 331 and 336 significantly altered or eliminated the modulatory effects of ethanol. Linear regression analyses revealed a significant relationship between hydropathy and polarity, but not molecular volume/molecular weight of the residues at these two positions. The results support the proposed hypothesis and represent an important step toward developing ethanol-insensitive receptors for investigating the role of P2X4Rs in mediating behavioral effects of ethanol.     

有机相中固定化脂肪酶促有机硅烷醇的转酯

探讨了有机相中固定化脂肪酶（Lipozyme）催化非天然的有机硅院醇与脂肪酸酯转酯反应的可能性;系统地研究了有机溶剂特性、水活度、有机硅烷醇结构、脂肪酸酯碳链长等因素对转酯反应的影响。     

Studies on Analogues of Succinic Semialdehyde as Substrates for Succinate Semialdehyde Dehydrogenase from Rat Brain

The methyl ester of succinic semialdehyde (SSA) was examined as a substrate for succinate semialdehyde dehydrogenase (SSADH) from rat brain. It was found that the ester can be oxidized by the enzyme. Values of Km for SSA-Me were higher than for those for SSA, and for this substrate the enzyme showed a substrate-dependent inhibition. This finding suggests that the carboxylate group of SSA is not essential in the process of inhibition of SSADH by the substrate. Cyclopropyl analogues of SSA, cis- and trans-1-formyl-cyclopropan-2-carboxylic acids, were also individually tested as substrates of SSADH. Only the trans isomer was found to be oxidized to the corresponding dicarboxylic acid; it inhibited the enzyme in the same range of concentrations as SSA. The above data suggest that, as for gamma-aminobutyric acid, SSA is present in an unfolded, transoid conformation at the active site of SSADH.     

一种快速提取质粒DNA用于鉴别重组克隆的方法

介绍了一种利用过夜培养的菌液瞬时提取质粒DNA,并用于电泳鉴别含有插入子克隆的方法。事先无需准备许多繁琐的相关试剂,提取质粒的全过程只需3~5min就可完成,非常适合于做重组克隆的快速鉴别。