Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands

Bilirubin oxidase (EC:1.3.3.5) purified from a culture medium of Myrothecium verrucaria MT-1 (authentic enzyme) catalyzes the oxidation of bilirubin to biliverdin in vitro and recombinant enzyme (wild type) was obtained by using an overexpression system of the bilirubin oxidase gene with Aspergillus oryzae harboring an expression vector. The absorption and ESR spectra showed that both bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers similar to laccase, ascorbate oxidase, and ceruloplasmin. Site-directed mutagenesis has been performed for the possible ligands of each type of copper. In some mutants, Cys457 --> Val, Ala, His94 --> Val, and His134.136 --> Val, type 1 and type 2 copper centers were perturbed completely and the enzyme activity was completely lost. Differing from the holoenzyme, these mutants showed type 3 copper signals. However, the optical and magnetic properties characteristic of type 1 copper were retained even by mutating one of the type 1 copper ligands, i.e., a mutant, Met467 --> Gly, showed a weak but apparent enzyme activity. A double mutant His456.458 --> Val had only type 1 Cu, showing a blue band at 600 nm (epsilon = 1.6 x 10(3)) and an ESR signal with very narrow hyperfine splitting (A parallel = 7.2 x 10(-)3 cm-1). Since the type 2 and type 3 coppers are not present, the mutant did not show enzyme activity. These results strongly imply that the peculiar sequence in bilirubin oxidase, His456-Cys457-His458, forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.     

Serum and urine metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common malignancy in the world with high morbidity and mortality rate. Identification of novel biomarkers in HCC remains impeded primarily because of the heterogeneity of the disease in clinical presentations as well as the pathophysiological variations derived from underlying conditions such as cirrhosis and steatohepatitis. The aim of this study is to search for potential metabolite biomarkers of human HCC using serum and urine metabolomics approach. Sera and urine samples were collected from patients with HCC (n = 82), benign liver tumor patients (n = 24), and healthy controls (n = 71). Metabolite profiling was performed by gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography-quadrupole time of flight mass spectrometry in conjunction with univariate and multivariate statistical analyses. Forty three serum metabolites and 31 urinary metabolites were identified in HCC patients involving several key metabolic pathways such as bile acids, free fatty acids, glycolysis, urea cycle, and methionine metabolism. Differentially expressed metabolites in HCC subjects, such as bile acids, histidine, and inosine are of great statistical significance and high fold changes, which warrant further validation as potential biomarkers for HCC. However, alterations of several bile acids seem to be affected by the condition of liver cirrhosis and hepatitis. Quantitative measurement and comparison of seven bile acids among benign liver tumor patients with liver cirrhosis and hepatitis, HCC patients with liver cirrhosis and hepatitis, HCC patients without liver cirrhosis and hepatitis, and healthy controls revealed that the abnormal levels of glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, and chenodeoxycholic acid are associated with liver cirrhosis and hepatitis. HCC patients with alpha fetoprotein values lower than 20 ng/ml was successfully differentiated from healthy controls with an accuracy of 100% using a panel of metabolite markers. Our work shows that metabolomic profiling approach is a promising screening tool for the diagnosis and stratification of HCC patients.     

Metabolomic profiling reveals bacterial metabolic adaptation strategies and new metabolites

How has metabolomics helped our understanding of infectious diseases? With the threat of antimicrobial resistance to human health around the world, metabolomics has emerged as a powerful tool to comprehensively characterize metabolic pathways to identify new drug targets. However, its output is constrained to known metabolites and their metabolic pathways. Recent advances in instrumentation, methodologies, and computational mass spectrometry have accelerated the use of metabolomics to understand pathogen–host metabolic interactions. This short review discusses a selection of recent publications using metabolomics in infectious/bacterial diseases. These studies unravel the links between metabolic adaptations to environments and host metabolic responses. Moreover, they highlight the importance of enzyme function and metabolite characterization in identifying new drug targets and biomarkers, as well as precision medicine in monitoring therapeutics and diagnosing diseases.     

Rescue of misrouted GnRHR mutants reveals its constitutive activity

G protein-coupled receptors (GPCR) play central roles in almost all physiological functions, and mutations in GPCR are responsible for over 30 hereditary diseases associated with loss or gain of receptor function. Gain of function mutants are frequently described as having constitutive activity (CA), that is, they activate effectors in the absence of agonist occupancy. Although many GPCR have mutants with CA, the GnRH receptor (GnRHR) was not, until 2010, associated with any CA mutants. The explanation for the failure to observe CA appears to be that the quality control system of the cell recognizes CA mutants of GnRHR as misfolded and retains them in the endoplasmic reticulum. In the present study, we identified several human (h)GnRHR mutants with substitutions in transmembrane helix 6 (F(272)K, F(272)Q, Y(284)F, C(279)A, and C(279)S) that demonstrate varying levels of CA after being rescued by pharmacoperones from different chemical classes and/or deletion of residue K(191), a modification that increases trafficking to the plasma membrane. The movement of the mutants from the endoplasmic reticulum (unrescued) to the plasma membrane (after rescue) is supported by confocal microscopy. Judging from the receptor-stimulated inositol phosphate production, mutants F(272)K and F(272)Q, after rescue, display the largest level of CA, an amount that is comparable with agonist-stimulated activation. Because mutations in other GPCR are, like the hGnRHR, scrutinized by the quality control system, this general approach may reveal CA in receptor mutants from other systems. A computer model of the hGnRHR and these mutants was used to evaluate the conformation associated with CA.     

从植物共生菌宏基因组文库筛选新的生物催化酶基因

【目的】通过建立宏基因组文库的高通量保存与基于探针洗脱的多次膜杂交筛选方法,从植物共生菌宏基因组文库筛选具有生物催化潜力的新酶基因。【方法】首先根据滴度将初始文库噬菌体包装颗粒感染到EPI300-T1R E.coli,过夜培养后对应保存于96孔板;提取粘粒进行文库的杂交筛选。【结果】描述的洗脱条件可完全去除尼龙膜上与靶DNA结合的探针,并且尼龙膜上的靶DNA至少可用于7次探针杂交,从而明显提高宏基因组文库的筛选效率。【结论】以Enoate reductase(ER)和短链脱氢酶(SDR)的同源基因片段为探针,运用该方法经两轮筛选获得候选单克隆并进行了部分粘粒的测序,发现了新的ER和SDR同源基因,并克隆到相应的全长基因序列用于后续的表达与酶化学研究。     

原玻璃蝇节杆菌D-氨基酸氧化酶及突变体的酶学特性

【目的】研究原玻璃蝇节杆菌(DSM 20168)中D-氨基酸氧化酶的酶学特性。【方法】通过PCR从原玻璃蝇节杆菌(DSM 15035,20168)中克隆获得D-氨基酸氧化酶基因apdaao-1和apdaao-2,构建原核表达载体,以表达质粒pET-ApDAAO-2为模板,采用QuickChange Site-Directed Mutagenesis技术构建定点突变体,经过原核表达及纯化获得重组型和突变体酶蛋白,分析其酶学特性。【结果】通过原核表达及纯化成功获得了2个重组蛋白和4个突变体酶蛋白,SDS-PAGE检测显示其分子量均约为36 kDa;酶学特性分析表明,ApDAAO-2和突变体蛋白的最适反应温度为30℃;ApDAAO-2和T286A的最适反应pH范围为7.0-11.0,其它突变体为8.0-11.0;ApDAAO-2和突变体都具有较广泛的底物特异性,除T256K的最适底物为D-Phe外,其余均为D-Met;动力学参数测定结果显示,以二级表观常数kcat/Km表示,对于底物D-Met或D-Phe,ApDAAO-2和4个突变体的kcat/Km值均比ApDAAO-1和pKDAAO高数倍以上。【结论】ApDAAO-2及突变体具有比ApDAAO-1和pKDAAO更广泛的底物特异性和较高的催化效率,有一定的商业应用价值。     

Purification and some properties of cyclohexylamine oxidase from a Pseudomonas sp.

Cyclohexylamine oxidase was purified 90-fold from cell-free extracts of Pseudomonas sp. capable of assimilating sodium cyclamate. The purified enzyme was homogeneous in disc electrophoresis, and the molecular weight was found to be approximately 80,000 by gel filtration. The enzyme catalyzed the following reaction: cyclohexylamine+O2+H2O leads to cyclohexanone+NH3+H2O2. The enzyme thus can be classified as an amine oxidase; it utilized oxygen as the ultimate electron acceptor. The pH optimum of the reaction was 6.8 and the apparent Km value for cyclohexylamine was 2.5 X 10(-4) M. The enzyme was highly specific for the deamination of alicyclic primary amines such as cyclohexylamine, but was found to be inactive toward ordinary amines used as substrates for amine oxidases. The enzyme solution was yellow in color and showed a typical flavoprotein spectrum; the addition of cyclohexylamine under anaerobic conditions caused reduction of the flavin in the native enzyme. The flavin of the prosthetic group was identified as FAD by thin layer chromatography. The participation of sulfhydryl groups in the enzymic action was also suggested by the observation that the enzyme activity was inhibited in the presence of PCMB and could be recovered by the addition of glutathione.     

唾液乳杆菌BSH1及其突变体的底物特异性

为了解析胆盐水解酶催化中心中关键氨基酸位点与其底物特异性的关系,以大肠杆菌pET-20b(+)表达系统为分子改造平台,采用理性设计,结合氨基酸定点突变的方法,成功构建了唾液乳杆菌Lactobacillus salivarius胆盐水解酶BSH1的7种突变体。通过对比L.salivarius BSH1及其突变体对6种结合胆盐的底物特异性表明,7种突变体对不同的结合胆盐的水解活性有所改变。结果说明,Cys2和Thr264分别是BSH1催化TCA和GCA的关键残基,且对酶的催化活性的保持具有关键作用。其中,高保守性的氨基酸位点Cys2不是BSH1唯一的活性位点,而其他突变的氨基酸位点可能作为BSH1的结合位点参与了底物的结合,也可能影响了底物进入BSH1活性中心的通道或底物结合口袋的体积与形状,进而影响了BSH1对不同结合胆盐的水解活性。     

Metabolite profiling reveals new insights into the regulation of serum urate in humans

Metabolomics - Serum urate, the final breakdown product of purine metabolism, is causally involved in the pathogenesis of gout, and implicated in cardiovascular disease and type 2 diabetes. Serum...     

Substrate inhibition of xanthine oxidase and its influence on superoxide radical production.

The influence of substrate inhibition on xanthine oxidase-intramolecular electron transport was studied by steady-state kinetic analysis. Experiments with hypoxanthine and xanthine up to 900 microM indicated an inhibition pattern which fitted an equation of the general form nu 0 = nu max . [S]/(Km + a[S] + b[S]2/Ki). Univalent electron flux to oxygen was favored at substrate concentrations above 50 microM. This augmentation of univalent flux percentage that appeared at a high substrate concentration was greater for hypoxanthine that xanthine and at pH 8.3 than at 9.5. Our results support a mechanism of inhibition in which a substrate-reduced enzyme, non-productive Michaelis complex was formed. It is possible that this non-productive complex favored the univalent pathway of enzyme reoxidation (superoxide production) by increasing the midpoint redox potential of the molybdenum active site.     

Enzymatic resolution of racemic amines in a continuous reactor in organic solvents

An enzymatic process has been developed for the continuous production of the pharmaceutically important intermediate (R)-1-aminoindan and of the chiral resolving agent (R)-1-(1-naphthyl)ethylamine. The process consists of the subtilisin catalyzed stereoselective aminolysis of the racemic primary amine with an active ester in organic solvent. The competing nonenzymatic reaction has been suppressed by appropriate choice of solvent and reactant's concentration and by minimizing the time of contact between the amine and the active ester. Subtilisin was immobilized on glass beads and the reaction carried out in a continuous-flow column bioreactor. By using a 450-mL column bioreactor containing 5.7 g of subtilisin immobilized on 570 g of glass beads, 1.6 kg of racemic 1-(1-naphthyl)ethylamine was resolved after 320 h of continuous operation with only a slight loss of the enzymatic activity. During the whole process, the optical purity of the chiral amine eluting from the column was higher than 90%. A facile procedure was developed for separating the unreacted (R)-amine from the (S)-amide and for the recycling of the solvent 3-methyl-3-pentanol and the active ester 2,2,2-trifluoroethyl butyrate. (c) 1992 John Wiley & Sons, Inc.     

Protein expression profiling of nuclear membrane protein reveals potential biomarker of human hepatocellular carcinoma

Background

Complex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes have been limited. Deciphering the molecular mechanism that differentiate between normal and disease state may lead to identification of biomarkers for carcinoma.

Results

Many proteins displayed differential expression when nuclear membrane proteome of hepatocellular carcinoma (HCC), fibrotic liver, and HepG2 cell line were assessed using 2-DE and ESI-Q-TOF MS/MS. From the down regulated set in HCC, we have identified for the first time a 15 KDa cytochrome b5A (CYB5A), ATP synthase subunit delta (ATPD) and Hemoglobin subunit beta (HBB) with 11, 5 and 22 peptide matches respectively. Furthermore, nitrosylation studies with S-nitrosocysteine followed by immunoblotting with anti SNO-cysteine demonstrated a novel and biologically relevant post translational modification of thiols of CYB5A in HCC specimens only. Immunofluorescence images demonstrated increased protein S-nitrosylation signals in the tumor cells and fibrotic region of HCC tissues. The two other nuclear membrane proteins which were only found to be nitrosylated in case of HCC were up regulated ATP synthase subunit beta (ATPB) and down regulated HBB. The decrease in expression of CYB5A in HCC suggests their possible role in disease progression. Further insight of the functional association of the identified proteins was obtained through KEGG/ REACTOME pathway analysis databases. String 8.3 interaction network shows strong interactions with proteins at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor of liver pathology.

Conclusion

These findings may have broader implications for understanding the mechanism of development of carcinoma. However, large scale studies will be required for further verification of their critical role in development and progression of HCC.     

Substrate profiling of PRMT1 reveals amino acid sequences that extend beyond the "RGG" paradigm

Protein arginine methyltransferase 1 (PRMT1) catalyzes the mono- and dimethylation of certain protein arginine residues. Although this posttranslational modification has been implicated in many physiological processes, the molecular basis for PRMT1 substrate recognition is poorly understood. Most modified arginine residues in known PRMT1 substrates reside in repeating "RGG" sequences. However, PRMT1 also specifically methylates Arg3 of histone H4 in a region that is not glycine-arginine rich, suggesting that PRMT1 substrates are not limited to proteins bearing "RGG" sequences. Because a systematic evaluation of PRMT1 substrate specificity has not been performed, it is unclear if the "RGG" sequence accurately represents the consensus target for PRMT1. Using a focused peptide library based on a sequence derived from the in vivo substrate fibrillarin we observed that PRMT1 methylated substrates that had amino acid residues other than glycine in the "RX (1)" and "RX (1)X (2)" positions. Importantly, eleven additional PRMT1 substrate sequences were identified. Our results also illustrate that the two residues on the N-terminal side of the modification site are important and need not both be glycine. PRMT1 methylated the eukaryotic initiation factor 4A1 (eIF4A1) protein, which has a single "RGG" sequence. Methylation of eIF4A1 and the similar eIF4A3 could be affected using single site mutations adjacent to the modification site, demonstrating the importance of amino acid sequence in PRMT1 protein substrates. Dimethylation of the parent library peptide was shown to occur through a dissociative mechanism. In summary, PRMT1 selectively recognizes a set of amino acid sequences in substrates that extend beyond the "RGG" paradigm.