定点突变提高虎杖聚酮合酶的苯亚甲基丙酮合酶活性北大核心CSCD

虎杖(Polygonum cuspidatum)聚酮合酶(polyketide synthase 1,PcPKS1)同时具有查尔酮合酶(chalcone synthase,CHS)及苯亚甲基丙酮合酶(benzylidene acetone synthase,BAS)催化活性,能够催化生成聚酮类化合物柚皮素查尔酮和苯亚甲基丙酮,进而催化合成黄酮类或覆盆子酮等具有多种生物学活性的化合物。本研究通过分析虎杖PcPKS1与掌叶大黄(Rheum palmatum)BAS、拟南芥(Arabidopsis thaliana)CHS等家族成员的序列以及酶催化位点的构象,确定可能影响酶功能的3个氨基酸位点:Thr133、Ser134、Ser339。采用定点突变对PcPKS1进行分子修饰,成功获得2个突变体并进行相关体外酶促反应,高效液相色谱(high performance liquid chromatography,HPLC)产物分析结果表明,在pH 7.0和pH 9.0的体外酶促条件下,突变体T133LS134A和S339V维持BAS和CHS双功能活性,且BAS活性显著高于原PcPKS1。本研究为利用PcPKS1进行基因工程调节黄酮类和覆盆子酮化合物的生物合成提供理论依据。     

Protection and reactivation of human methylmalonyl-CoA mutase by MMAA protein

Previous studies have reported that some adenosylcobalamin-dependent enzymes suffer inactivation during catalysis due to the oxidation of cobalamin. In addition, the protection or reactivation of their catalytic activities by proteins called “protectases” or reactivases is well known in bacteria. In this study, we examined the influence of human MMAA protein on the kinetics of the reaction catalyzed by methylmalonyl-CoA mutase (MCM) by testing both purified recombinant proteins in vitro. Our results showed that MMAA plays dual roles in MCM activity. When it was added at the beginning of the reaction, it prevents inactivation by guarding MCM. After 60 min of reaction, when MCM is inactive, the addition of MMAA increases the enzymatic activity through GTP hydrolysis, indicating reactivation of MCM by exchange of the damaged cofactor. Interaction between MCM and MMAA observed in vitro was confirmed in vivo by yeast two-hybrid system.     

High-performance liquid chromatographic and tandem mass spectrometric quantitation ofN7-methyldeoxyguanosine in methylated calf thymus DNA

Quantitation ofN7-methyldeoxyguanosine (N7-MedG) produced in thein vitro N-methyl-N-introsourea (NMU) action on calf thymus DNA has been achieved by enzymatic degradation, liquid chromatographic separation and desorption chemical ionization tandem mass spectrometry. In conjunction with the resolving power of HPLC in the separation of isomers, desorption chemical ionization tandem mass spectrometry has been utilized in determining modified nucleosides at low levels using a stable-isotope labeled compound as an established by an independent HPLC analysis of methylated calf thymus DNA. A sensitive and specific methodology for the quantitation ofN7-MedG at the picomole level using HPLC combined with tandem mass spectrometry without radioisotope labeling process is presented. The potential of the liquid chromatographic tandem mass spectrometric analysis shows the detection ofN7-MedG as a possible marker for human exposure to methylating agentsin vitro.     

Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase

Improvement of enzyme function by engineering pH dependence of enzymatic activity is of importance for industrial application of Bacillus circulans xylanases. Target mutation sites were selected by structural alignment between B. circulans xylanase and other xylanases having different pH optima. We selected non-conserved mutant sites within 8 Å from the catalytic residues, to see whether these residues have some role in modulating pK_as of the catalytic residues. We hypothesized that the non-conserved residues which may not have any role in enzyme catalysis might perturb pK_as of the catalytic residues. Change in pK_a of a titratable group due to change in electrostatic potential of a mutation was calculated and the change in pH optimum was predicted from the change in pK_a of the catalytic residues. Our strategy is proved to be useful in selection of promising mutants to shift the pH optimum of the xylanases towards desired side.     

Detoxification of G- and V-series nerve agents by the phosphotriesterase OpdA

Abstract

Intoxication by organophosphorous (OP) insecticides and nerve agents is often lethal and currently available therapeutics are often ineffective. A range of catalytic and stoichiometric OP scavengers have been investigated for use as potential treatments for OP poisoning. Recent studies have shown that one enzyme, OpdA, an enzyme involved in organophosphorous degradation, was an effective treatment for OP insecticide poisoning in animal models. Here we have tested OpdA for its ability to detoxify G- and V-type nerve agents in vitro. Although OpdA was found to have high catalytic activities for G-series toxins (soman and cyclosarin), it was substantially less active with V-type nerve agents. The activity towards V-series agents was close to the theoretical maximum for this enzyme (i.e. the rate determined by the chemistry of the leaving group); it seems unlikely that enzyme engineering or directed evolution could be used to improve upon this activity without a significant change in its reaction mechanism.     

Expression and purification of the D4 region of PLD1 and characterization of its interaction with PED-PEA15

PLD’s (Phospholipases D) are ubiquitously expressed proteins involved in many transphosphatidylation reactions. They have a bi-lobed structure composed by two similar domains which at their interface reconstitute the catalytic site through the association of the two conserved HxKx₄Dx₆GSxN motifs. PLD1 interacts with the small phosphoprotein PED-PEA15 by an unknown mechanism that, by enhancing PLD1 stability, apparently increases its enzymatic activity; the minimum interacting region of PLD1 was previously identified as spanning residues 712–1074 (D4 region). Since the D4/PED-PEA15 interaction has been claimed to be one of the multiple molecular events that can trigger type 2 diabetes, we purified the two recombinant proteins to study in vitro this binding by both ELISA and SPR techniques. Whilst PED-PEA15 was easily expressed and purified, expression of recombinant D4 was more problematic and only the fusion protein with Thioredoxin A and a six Histidine Tag (Trx-His₆-D4) demonstrated sufficient stability for further characterization. We have found that Trx-His₆-D4 is present as two different oligomeric forms, though only the monomeric variant is able to interact with PED-PEA15. All these findings may have important implications for both the mechanisms of phospholipase activity and PED-PEA15 regulative functions.     

Structure,mechanism, and enantioselectivity shifting of lipase LipK107 with a simple way

Because of the complex mechanisms of enzymatic reactions, no precise and simple method of understanding and controlling the chiral selectivity of enzymes has been developed. However, structure-based rational design is a powerful approach to engineering enzymes with desired catalytic activities. In this work, a simple, structure-based, large-scale in silico design and virtual screening strategy was developed and successfully applied to enzyme engineering. We first performed protein crystallization and X-ray diffraction to determine the structure of lipase LipK107, which is a novel family I.1 lipase displaying activity for both R and S isomers in chiral resolution reactions. The catalytic mechanism of family I.1, which includes LipK107, was ascertained first through comparisons of the sequences and structures of lipases from other families. The binding states of LipK107, including the energy and the conformation of complexes with the R and S enantiomers, have been evaluated by careful biocomputation to figure out the reason for the higher S selectivity. Based on this study, a simple strategy for manipulating the chiral selectivity by modulating a crucial distance in the enzyme–substrate complex and judging virtual mutations in silico is recommended. Then, a novel electrostatic interaction analysis protocol was used to design LipK107 mutants to validate our strategy. Both positive and negative mutations determined using this theoretical protocol have been implemented in wet experiments and were proved to produce the desired enantioselectivity, showing a 176% increase or 50% decrease in enantioselectivity as desired. Because of its accuracy and versatility, the strategy is promising for practical applications.     

CarbonCarbon Bond Formation by Lewis Superacid Catalysis

Diverse intramolecular cyclizations involving the formation of C? C bonds are described using catalytic methodologies based on Lewis superacids. Examples are presented on 1,6‐diene cyclizations to gem‐dimethylcyclohexane structures. Tandem cyclization of trienes are described to afford bicyclic structures in reactions involving rearrangements. Hydroarylation of olefins and of allenes is developed in catalytic Friedel? Crafts‐type coupling processes, which can give rise to tandem reactions. The olfactory evaluation of the series of prepared compounds is also presented.     

Systematic analysis of T7 RNA polymerase basedin vitrolinear RNA amplification for use in microarray experiments

Background  

The requirement of a large amount of high-quality RNA is a major limiting factor for microarray experiments using biopsies. An average microarray experiment requires 10–100 μg of RNA. However, due to their small size, most biopsies do not yield this amount. Several different approaches for RNA amplificationin vitrohave been described and applied for microarray studies. In most of these, systematic analyses of the potential bias introduced by the enzymatic modifications are lacking.     

Clinical Use of Aromatase Inhibitors: Current Data and Future Perspectives

Abstract

A systematic procedure for the kinetic study of irreversible inhibition when the enzyme is consumed in the reaction which it catalyses, has been developed and analysed. Whereas in most reactions the enzymes are regenerated after each catalytic event and serve as reusable transacting effectors, in the consumed enzymes each catalytic center participates only once and there is no enzyme turnover. A systematic kinetic analysis of irreversible inhibition of these enzyme reactions is presented. Based on the algebraic criteria proposed in this work, it should be possible to evaluate either the mechanism of inhibition (complexing or non-complexing), or the type of inhibition (competitive, non-competitive, uncompetitive, mixed non-competitive). In addition, all kinetic constants involved in each case could be calculated. An experimental application of this analysis is also presented, concerning peptide bond formation in vitro. Using the puromycin reaction, which is a model reaction for the study of peptide bond formation in vitro and which follows the same kinetic law as the enzymes under study, we have found that: (i) the antibiotic spiramycin inhibits the puromycin reaction as a competitive irreversible inhibitor in a one step mechanism with an association rate constant equal to 1.3 × 10⁴M^-1s^-1 and, (ii) hydroxylamine inhibits the same reaction as an irreversible non-competitive inhibitor also in a one step mechanism with a rate constant equal to 1.6 × 10^-3 M^-1s^-1.     

Identification of methylated flavonoid regioisomeric metabolites using enzymatic semisynthesis and liquid chromatography-tandem mass spectrometry

Discrimination of isomeric methylated metabolites is an important step toward identifying genes responsible for methylation, but presents substantial challenges because authentic standards are often unavailable and mass spectra of isomers have been considered indistinguishable. In this report, an approach is described for identifying methyl group positions in multiply methylated flavonoid metabolites using combinations of tandem mass spectrometry, liquid chromatography retention, and site-selective methylation by recombinant O-methyltransferases from Solanum habrochaites LA1777. The basis for observed fragment ions in tandem mass spectra of multiply methylated myricetin was further established using enzymatic incorporation of deuterium-labeled methyl groups using S-adenosylmethionine-d ₃ as precursor.

     

The histochemical demonstration of aniline hydroxylase activity in rat liver

Synopsis A procedure is described for the histochemical demonstration of aniline hydroxylase activity in cryostat sections of rat liver. Tissue sections are incubated in a medium containing aniline; thep-aminophenol formed as a result of enzymatic action is coupledin situ with Fast Blue RR. The staining reaction is found to be confined to the cytoplasm of the hepatocytes. Confirmatory tests for true enzymatic staining reaction include the incubation of sections in medium from which aniline is omitted, and under conditions of enzyme inhibibition. A method for the quantitation of the histochemical staining reaction is also described.The histochemical reactions have been investigated on rat livers subjected to conditions eliciting microsomal enzyme stimulation and inhibition, bothin vitro andin vivo. A close correlation was found between the staining reactions observed and the results of the quantitative histochemical method and the biochemical estimations of aniline hydroxylase activity in liver microsomal fractions obtained by differential centrifugation.     

Bacterial mandelic acid degradation pathway and its application in biotechnology

Mandelic acid and its derivatives are an important class of chemical synthetic blocks, which is widely used in drug synthesis and stereochemistry research. In nature, mandelic acid degradation pathway has been widely identified and analysed as a representative pathway of aromatic compounds degradation. The most studied mandelic acid degradation pathway from Pseudomonas putida consists of mandelate racemase, S-mandelate dehydrogenase, benzoylformate decarboxylase, benzaldehyde dehydrogenase and downstream benzoic acid degradation pathways. Because of the ability to catalyse various reactions of aromatic substrates, pathway enzymes have been widely used in biocatalysis, kinetic resolution, chiral compounds synthesis or construction of new metabolic pathways. In this paper, the physiological significance and the existing range of the mandelic acid degradation pathway were introduced first. Then each of the enzymes in the pathway is reviewed one by one, including the researches on enzymatic properties and the applications in biotechnology as well as efforts that have been made to modify the substrate specificity or improving catalytic activity by enzyme engineering to adapt different applications. The composition of the important metabolic pathway of bacterial mandelic acid degradation pathway as well as the researches and applications of pathway enzymes is summarized in this review for the first time.     

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis

BackgroundAnalysis of limiting steps within enzyme-catalyzed reactions is fundamental to understand their behavior and regulation. Methods capable of unravelling control properties and exploring kinetic capabilities of enzymatic reactions would be particularly useful for protein and metabolic engineering. While single-enzyme control analysis formalism has previously been applied to well-studied enzymatic mechanisms, broader application of this formalism is limited in practice by the limited amount of kinetic data and the difficulty of describing complex allosteric mechanisms.MethodsTo overcome these limitations, we present here a probabilistic framework enabling control analysis of previously unexplored mechanisms under uncertainty. By combining a thermodynamically consistent parameterization with an efficient Sequential Monte Carlo sampler embedded in a Bayesian setting, this framework yields insights into the capabilities of enzyme-catalyzed reactions with modest kinetic information, provided that the catalytic mechanism and a thermodynamic reference point are defined.ResultsThe framework was used to unravel the impact of thermodynamic affinity, substrate saturation levels and effector concentrations on the flux control and response coefficients of a diverse set of enzymatic reactions.ConclusionsOur results highlight the importance of the metabolic context in the control analysis of isolated enzymes as well as the use of statistically sound methods for their interpretation.General SignificanceThis framework significantly expands our current capabilities for unravelling the control properties of general reaction kinetics with limited amount of information. This framework will be useful for both theoreticians and experimentalists in the field.     

The metabolic core and catalytic switches are fundamental elements in the self-regulation of the systemic metabolic structure of cells

Background

Experimental observations and numerical studies with dissipative metabolic networks have shown that cellular enzymatic activity self-organizes spontaneously leading to the emergence of a metabolic core formed by a set of enzymatic reactions which are always active under all environmental conditions, while the rest of catalytic processes are only intermittently active. The reactions of the metabolic core are essential for biomass formation and to assure optimal metabolic performance. The on-off catalytic reactions and the metabolic core are essential elements of a Systemic Metabolic Structure which seems to be a key feature common to all cellular organisms.

Methodology/Principal Findings

In order to investigate the functional importance of the metabolic core we have studied different catalytic patterns of a dissipative metabolic network under different external conditions. The emerging biochemical data have been analysed using information-based dynamic tools, such as Pearson''s correlation and Transfer Entropy (which measures effective functionality). Our results show that a functional structure of effective connectivity emerges which is dynamical and characterized by significant variations of bio-molecular information flows.

Conclusions/Significance

We have quantified essential aspects of the metabolic core functionality. The always active enzymatic reactions form a hub –with a high degree of effective connectivity- exhibiting a wide range of functional information values being able to act either as a source or as a sink of bio-molecular causal interactions. Likewise, we have found that the metabolic core is an essential part of an emergent functional structure characterized by catalytic modules and metabolic switches which allow critical transitions in enzymatic activity. Both, the metabolic core and the catalytic switches in which also intermittently-active enzymes are involved seem to be fundamental elements in the self-regulation of the Systemic Metabolic Structure.     

On the importance of orientation in general base catalysis by carboxylate

The carboxylate group serves as a general-base catalyst in numerous chemical and enzymatic reactions. The importance of the direction in which the proton is transferred to the carboxylate is discussed. syn (on the same side of the CO bond as the forming CO) protonation is estimated to be 10⁴-fold more favorable than anti protonation. To date, in the models studied where intramolecular reactions involve carboxylate, only anti protonation can occur. In these intramolecular models the catalytic efficiency of a carboxylate group may be underestimated due to this inability to achieve an optimal orientation for protonation; whereas for enzymatic reactions involving carboxylate side chains, structural studies support mechanisms involving syn protonation.     

Designer metalloenzymes for synthetic biology: Enzyme hybrids for catalysis

Combining organometallics and biology has generated broad interest from scientists working on applications from in situ drug release to biocatalysis. Engineered enzymes and biohybrid catalysts (also referred to as artificial enzymes) have introduced a wide range of abiotic chemistry into biocatalysis. Predominantly, this work has concentrated on using these catalysts for single step in vitro reactions. However, the promise of using these hybrid catalysts in vivo and combining them with synthetic biology and metabolic engineering is vast. This report will briefly review recent advances in artificial metalloenzyme design, followed by summarising recent studies that have looked at the use of these hybrid catalysts in vivo and in enzymatic cascades, therefore exploring their potential for synthetic biology.     

Systematic phenome analysis of Escherichia coli multiple‐knockout mutants reveals hidden reactions in central carbon metabolism

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple‐knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose‐7‐phosphate to erythrose‐4‐phosphate and dihydroxyacetone phosphate were observed in transaldolase‐deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose‐7‐phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP‐dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.