Chemical Synthesis of Trans 8-Methyl-6-Nonenoyl-CoA and Functional Expression Unravel Capsaicin Synthase Activity Encoded by the Pun1 Locus

Capsaicin, produced by diverse Capsicum species, is among the world’s most popular spices and of considerable pharmaceutical relevance. Although the capsaicinoid biosynthetic pathway has been investigated for decades, several biosynthetic steps have remained partly hypothetical. Genetic evidence suggested that the decisive capsaicin synthase is encoded by the Pun1 locus. Yet, the genetic evidence of the Pun1 locus was never corroborated by functionally active capsaicin synthase that presumably catalyzes an amide bond formation between trans 8-methyl-6-nonenoyl-CoA derived from branched-chain amino acid biosynthesis and vanilloylamine derived from the phenylpropanoid pathway. In this report, we demonstrate the enzymatic activity of a recombinant capsaicin synthase encoded by Pun1, functionally expressed in Escherichia coli, and provide information on its substrate specificity and catalytic properties. Recombinant capsaicin synthase is specific for selected aliphatic CoA-esters and highly specific for vanilloylamine. Partly purified from E. coli, the recombinant active enzyme is a monomeric protein of 51 kDa that is independent of additional co-factors or associated proteins, as previously proposed. These data can now be used to design capsaicin synthase variants with different properties and alternative substrate preferences.     

PeSA: A software tool for peptide specificity analysis

The discovery and characterization of molecular interactions is crucial towards a better understanding of complex biological processes. Particularly protein-protein interactions (i.e., PPIs), which are responsible for a variety of cellular functions from intracellular signaling to enzyme-substrate specificity, have been studied broadly over the past decades. Position-specific scoring matrices (PSSM) in particular are used extensively to help determine interaction specificity or candidate interaction motifs at the residue level. However, not all studies successfully report their results as a candidate interaction motif. In many cases, this may be due to a lack of suitable tools for simple analysis and motif generation. Peptide Specificity Analyst (PeSA) was developed with the goal of filling this information gap and providing an easy to use software to aid peptide array analysis and motif generation. PeSA utilizes two models of motif creation: (1) frequency-based using a user-defined peptide list, and (2) weight-based using experimental binding results. The ability to produce motifs effortlessly will make studying, interpreting and disseminating peptide specificity results in an effortless and straightforward process.     

OH+H2S→H2O+SH反应的模式特异性量子动力学研究

研究氢抽取反应OH+H₂S对于理解酸雨形成、空气污染和气候变化的原因具有重要意义. 本文在降维模型下使用量子含时波包方法研究了OH+H₂S→H₂O+SH反应的动力学行为. 研究表明，该反应在低碰撞能时表现出无垒反应的特征，而在高碰撞能下表现出具有显著势垒的激活反应的特征. 激发反应物H₂S分子的对称或反对称伸缩模式比激发弯曲模式更有效地促进了反应，该动力学特征可以通过各正则模式与反应坐标的耦合强度来解释. 此外，模式指定的反应速率常数表现出明显的非阿伦尼乌斯温度依赖性.     

Composition analysis of positional isomers of phosphatidylinositol by high-performance liquid chromatography

An HPLC-based method has been developed for composition analysis of six positional isomers of phosphatidylinositol (PI), of which the phosphatidyl group was connected to different positions of the myo-inositol moiety. The method employed a combination of two types of HPLC analyses. One was direct separation of the six PI isomers into four peaks of 1(3)-PI, 2-PI, 4(6)-PI and 5-PI on a normal-phase silica gel column. The second method was for the separations of 1-PI from 3-PI and 4-PI from 6-PI, which were not separable on the normal-phase column. This method involved conversion of PI isomers into pentakis-(R)-1-phenylethylcarbamate (PEC) derivatives, which were separated on a reversed-phase column. Using the established method, positional specificity of several engineered phospholipases D in enzymatic synthesis of PI from myo-inositol and phosphatidylcholine was investigated. This was performed by analyzing the isomeric composition of PIs synthesized by the mutant enzymes. Among five mutant enzymes tested, two showed strong specificity to 1-OH, one showed moderate preference to 1-OH, one preferred 3-OH, and one showed broad specificity towards 1-, 3-, 4- and 6-OH.     

Using Steady-State Kinetics to Quantitate Substrate Selectivity and Specificity: A Case Study with Two Human Transaminases

We examined the ability of two human cytosolic transaminases, aspartate aminotransferase (GOT1) and alanine aminotransferase (GPT), to transform their preferred substrates whilst discriminating against similar metabolites. This offers an opportunity to survey our current understanding of enzyme selectivity and specificity in a biological context. Substrate selectivity can be quantitated based on the ratio of the k_cat/K_M values for two alternative substrates (the ‘discrimination index’). After assessing the advantages, implications and limits of this index, we analyzed the reactions of GOT1 and GPT with alternative substrates that are metabolically available and show limited structural differences with respect to the preferred substrates. The transaminases’ observed selectivities were remarkably high. In particular, GOT1 reacted ~10⁶-fold less efficiently when the side-chain carboxylate of the ’physiological’ substrates (aspartate and glutamate) was replaced by an amido group (asparagine and glutamine). This represents a current empirical limit of discrimination associated with this chemical difference. The structural basis of GOT1 selectivity was addressed through substrate docking simulations, which highlighted the importance of electrostatic interactions and proper substrate positioning in the active site. We briefly discuss the biological implications of these results and the possibility of using k_cat/K_M values to derive a global measure of enzyme specificity.     

Substrate Specificity of Chimeric Enzymes Formed by Interchange of the Catalytic and Specificity Domains of the 5′-Nucleotidase UshA and the 3′-Nucleotidase CpdB

The 5′-nucleotidase UshA and the 3′-nucleotidase CpdB from Escherichia coli are broad-specificity phosphohydrolases with similar two-domain structures. Their N-terminal domains (UshA_Ndom and CpdB_Ndom) contain the catalytic site, and their C-terminal domains (UshA_Cdom and CpdB_Cdom) contain a substrate-binding site responsible for specificity. Both enzymes show only partial overlap in their substrate specificities. So, it was decided to investigate the catalytic behavior of chimeras bearing the UshA catalytic domain and the CpdB specificity domain, or vice versa. UshA_Ndom–CpdB_Cdom and CpdB_Ndom–UshA_Cdom were constructed and tested on substrates specific to UshA (5′-AMP, CDP-choline, UDP-glucose) or to CpdB (3′-AMP), as well as on 2′,3′-cAMP and on the common phosphodiester substrate bis-4-NPP (bis-4-nitrophenylphosphate). The chimeras did show neither 5′-nucleotidase nor 3′-nucleotidase activity. When compared to UshA, UshA_Ndom–CpdB_Cdom conserved high activity on bis-4-NPP, some on CDP-choline and UDP-glucose, and displayed activity on 2′,3′-cAMP. When compared to CpdB, CpdB_Ndom–UshA_Cdom conserved phosphodiesterase activities on 2′,3′-cAMP and bis-4-NPP, and gained activity on the phosphoanhydride CDP-choline. Therefore, the non-nucleotidase activities of UshA and CpdB are not fully dependent on the interplay between domains. The specificity domains may confer the chimeras some of the phosphodiester or phosphoanhydride selectivity displayed when associated with their native partners. Contrarily, the nucleotidase activity of UshA and CpdB depends strictly on the interplay between their native catalytic and specificity domains.     

Identification of degradation impurities in aripiprazole oral solution using LC–MS and development of validated stability indicating method for assay and content of two preservatives by RP-HPLC

A simple and simultaneous reverse phase high-performance liquid chromatographic method was developed for the quantification of aripiprazole (ARI) and two preservatives, namely, methyl paraben and propyl paraben in ARI oral solution. The method was developed on ACE C18 (4.6?×?250?mm, 5?µm) column using gradient elution of 0.1% v/v trifluoroacetic acid in water and acetonitrile as mobile phase components. Flow rate of 1.0?mL/min and 30°C column temperature were used for the method at quantification wavelength of 254?nm. The developed method was validated in accordance with International Conference on Harmonization guideline for various parameters. Forced degradation study was conducted in acid, base, peroxide, heat, and light stress conditions. ARI was found to degrade in oxidation, acid hydrolysis, and heat while it was stable under the remaining conditions. Specificity of the method was verified using Photo Diode Array (PDA) detector by evaluating purity of peaks from degradation samples. Major degradation impurities formed during stress study were identified using liquid chromatography–mass spectrometry method. The present method was useful for determining the content of all the three main analytes present in the oral solution without interference from degradation impurities. The method was robust under the deliberately modified conditions.     

Structural Elucidation of the Bispecificity of A Domains as a Basis for Activating Non‐natural Amino Acids

Many biologically active peptide secondary metabolites of bacteria are produced by modular enzyme complexes, the non‐ribosomal peptide synthetases. Substrate selection occurs through an adenylation (A) domain, which activates the cognate amino acid with high fidelity. The recently discovered A domain of an Anabaenopeptin synthetase from Planktothrix agardhii (ApnA A₁) is capable of activating two chemically distinct amino acids (Arg and Tyr). Crystal structures of the A domain reveal how both substrates fit into to binding pocket of the enzyme. Analysis of the binding pocket led to the identification of three residues that are critical for substrate recognition. Systematic mutagenesis of these residues created A domains that were monospecific, or changed the substrate specificity to tryptophan. The non‐natural amino acid 4‐azidophenylalanine is also efficiently activated by a mutant A domain, thus enabling the production of diversified non‐ribosomal peptides for bioorthogonal labeling.     

可编程序控制器系统的可靠性数学模型和应用

本文提出了可编程序控制器系统的可靠性数学模型，并应用该模型讨论了可编程序控制器系统的故障特性，最后给出了提高系统可靠性的方法。