The kinetic model of the shikimate pathway as a tool to optimize enzyme assays for high-throughput screening

Four-enzyme section of the shikimate pathway (Aro B, D, E, and K) of Streptococcus pneumoniae has been studied. Kinetic properties of the individual enzymes and three- and four-enzyme linked reactions have been characterized in vitro. On the basis of the data measured in spectrophotometric and LC-MS experiments, kinetic mechanisms of the enzymes have been suggested and all kinetic parameters have been identified. Kinetic models for these three- and four-enzyme sections of the shikimate pathway have been constructed and validated. The model of the four-enzyme section of shikimate pathway has been employed to design an inhibition-sensitive reconstituted pathway for a high-throughput screening effort on the shikimate pathway. It was demonstrated that using the model it was possible to optimize this reconstituted pathway in such a way to provide equal sensitivity of the enzymes to inhibition.     

Post-translational control of the long and winding road to cholesterol

The synthesis of cholesterol requires more than 20 enzymes, many of which are intricately regulated. Post-translational control of these enzymes provides a rapid means for modifying flux through the pathway. So far, several enzymes have been shown to be rapidly degraded through the ubiquitin–proteasome pathway in response to cholesterol and other sterol intermediates. Additionally, several enzymes have their activity altered through phosphorylation mechanisms. Most work has focused on the two rate-limiting enzymes: 3-hydroxy-3-methylglutaryl CoA reductase and squalene monooxygenase. Here, we review current literature in the area to define some common themes in the regulation of the entire cholesterol synthesis pathway. We highlight the rich variety of inputs controlling each enzyme, discuss the interplay that exists between regulatory mechanisms, and summarize findings that reveal an intricately coordinated network of regulation along the cholesterol synthesis pathway. We provide a roadmap for future research into the post-translational control of cholesterol synthesis, and no doubt the road ahead will reveal further twists and turns for this fascinating pathway crucial for human health and disease.     

保幼激素生物合成研究进展

保幼激素（juvenile hormone,JH）是存在于昆虫、甲壳动物和部分植物体内的倍半萜类衍生物。在昆虫和甲壳动物体内,保幼激素主要调节变态和生殖活动。在植物体内,则可能作为异株克生物质发挥作用。保幼激素主要通过细胞质内的甲羟戊酸途径（MVA）合成,植物质体内存在萜类合成的1-去氧木糖-5-磷酸途径（DXP）。MVA和DXP途径通过单向质子协同运输系统进行协调,使DXP途径中形成的前体化合物参与MVA途径的倍半萜合成。JH生物合成的主要步骤己基本查明,但与合成相关的酶学研究还较薄弱。生物合成酶的分子生物学是近来研究的热点,相关酶的cDNA克隆已有报道。JH生物合成酶的进一步研究有助于查明JH生物合成调控机制,深化对节肢动物生殖的理解,还可为新型杀虫剂开发提供可能的靶标。     

Closing in on complete pathways of biotin biosynthesis

Biotin is an enzyme cofactor indispensable to metabolic fixation of carbon dioxide in all three domains of life. Although the catalytic and physiological roles of biotin have been well characterized, the biosynthesis of biotin remains to be fully elucidated. Studies in microbes suggest a two-stage biosynthetic pathway in which a pimelate moiety is synthesized and used to begin assembly of the biotin bicyclic ring structure. The enzymes involved in the bicyclic ring assembly have been studied extensively. In contrast the synthesis of pimelate, a seven carbon α,ω-dicarboxylate, has long been an enigma. Support for two different routes of pimelate synthesis has recently been obtained in Escherichia coli and Bacillus subtilis. The E. coli BioC-BioH pathway employs a methylation and demethylation strategy to allow elongation of a temporarily disguised malonate moiety to a pimelate moiety by the fatty acid synthetic enzymes whereas the B. subtilis BioI-BioW pathway utilizes oxidative cleavage of fatty acyl chains. Both pathways produce the pimelate thioester precursor essential for the first step in assembly of the fused rings of biotin. The enzymatic mechanisms and biochemical strategies of these pimelate synthesis models will be discussed in this review.     

The molecular architecture of major enzymes from ajmaline biosynthetic pathway

The biosynthetic pathway leading to the monoterpenoid indole alkaloid ajmaline in Rauvolfia serpentiin serpentina is one of the most studied in the field of natural product biosynthesis. Ajmaline has a complex structure which is based on a six-membered ring system harbouring nine chiral carbon atoms. There are about fifteen enzymes involved, including some involving the side reactions of the ajmaline biosynthetic pathway. All enzymes exhibit pronounced substrate specificity. In the recent years isolation and sequencing of their cDNAs has allowed a detailed sequence analysis and comparison with functionally related and occasionally un-related enzymes. Site-directed mutations of several of the ajmaline-synthesizing enzymes have been performed and their catalytic residues have been identified. Success with over-expression of the enzymes was an important step for their crystallization and structural analysis by X-ray crystallography. Crystals with sufficient resolution were obtained from the major enzymes of the pathway. Strictosidine synthase has a 3D-structure with a six-bladed β-propeller fold the first time such a fold found in the plant kingdom. Its ligand complexes with tryptamine and secologanin, as well as structure-based sequence alignment, indicate a possible evolutionary relationship to several primary sequence-unrelated structures with this fold. The structure of strictosidine glucosidase was determined and its structure has as a (β/α)₈ barrel fold. Vinorine synthase provides the first 3D structure of a member of BAHD enzyme super-family. Raucaffricine glucosidase involved in a side-route of ajmaline biosynthesis has been crystallized. The ajmaline biosynthetic pathway is an outstanding example where many enzymes 3D-structure have been known and where there is a real potential for protein engineering to yield new alkaloid.     

The folic acid biosynthesis pathway in bacteria: evaluation of potential for antibacterial drug discovery

The potential of the folic acid biosynthesis pathway as a target for the development of antibiotics has been acknowledged for many years and validated by the clinical use of several drugs. Recently, the crystal structures of all but one of the enzymes in the pathway from GTP to dihydrofolate have been determined. Given that structure-based drug design strategies are now widely employed, these recent developments have prompted a re-evaluation of the potential of each of the enzymes in the pathway as a target for development of specific inhibitors. Here, we review the current knowledge of the structure and mechanism of each enzyme in the bacterial folic acid biosynthesis pathway from GTP to dihydrofolate and draw conclusions regarding the potential of each enzyme as a target for therapeutic intervention.     

Chemical and genomic evolution of enzyme-catalyzed reaction networks

There is a tendency that a unit of enzyme genes in an operon-like structure in the prokaryotic genome encodes enzymes that catalyze a series of consecutive reactions in a metabolic pathway. Our recent analysis shows that this and other genomic units correspond to chemical units reflecting chemical logic of organic reactions. From all known metabolic pathways in the KEGG database we identified chemical units, called reaction modules, as the conserved sequences of chemical structure transformation patterns of small molecules. The extracted patterns suggest co-evolution of genomic units and chemical units. While the core of the metabolic network may have evolved with mechanisms involving individual enzymes and reactions, its extension may have been driven by modular units of enzymes and reactions.     

The ins and outs of sphingolipid synthesis

Sphingolipids are ubiquitous components of eukaryotic cell membranes, where they play important roles in intracellular signaling and in membrane structure. Even though the biochemical pathway of sphingolipid synthesis and its compartmentalization between the endoplasmic reticulum and Golgi apparatus have been known for many years, the molecular identity of the enzymes in this pathway has only recently been elucidated. Here, we summarize progress in the identification and characterization of the enzymes, the transport of ceramide from the endoplasmic reticulum to the Golgi apparatus, and discuss how regulating the synthesis of sphingolipids might impact upon their functions.     

Post-translational control of tetrapyrrole biosynthesis in plants, algae, and cyanobacteria

The tetrapyrrole biosynthetic pathway provides the vital cofactors and pigments for photoautotrophic growth (chlorophyll), several essential redox reactions in electron transport chains (haem), N- and S-assimilation (sirohaem), and photomorphogenic processes (phytochromobilin). While the biochemistry of the pathway is well understood and almost all genes encoding enzymes of tetrapyrrole biosynthesis have been identified in plants, the post-translational control and organization of the pathway remains to be clarified. Post-translational mechanisms controlling metabolic activities are of particular interest since tetrapyrrole biosynthesis needs adaptation to environmental challenges. This review surveys post-translational mechanisms that have been reported to modulate metabolic activities and organization of the tetrapyrrole biosynthesis pathway.     

Structural enzymology of biotin biosynthesis

Over the last years, significant progress has been made in the understanding of the genetics and enzymology of the biosynthetic pathway of the vitamin biotin. The enzymes catalyzing the last four steps of this pathway, from pimeloyl-CoA to biotin, provide an ensemble of intriguing reaction mechanisms, which are presently being unravelled. The three-dimensional structures for three of these enzymes are known and provide a framework to which on-going mechanistic studies can be related.     

Processing of a class I-restricted epitope from tyrosinase requires peptide N-glycanase and the cooperative action of endoplasmic reticulum aminopeptidase 1 and cytosolic proteases

Although multiple components of the class I MHC processing pathway have been elucidated, the participation of nonproteasomal cytosolic enzymes has been largely unexplored. In this study, we provide evidence for multiple cytosolic mechanisms in the generation of an HLA-A*0201-associated epitope from tyrosinase. This epitope is presented in two isoforms containing either Asn or Asp, depending on the structure of the tyrosinase precursor. We show that deamidation of Asn to Asp is dependent on glycosylation in the endoplasmic reticulum (ER), and subsequent deglycosylation by peptide-N-glycanase in the cytosol. Epitope precursors with N-terminal extensions undergo a similar process. This is linked to an inability of ER aminopeptidase 1 to efficiently remove N-terminal residues, necessitating processing by nonproteasomal peptidases in the cytosol. Our work demonstrates that processing of this tyrosinase epitope involves recycling between the ER and cytosol, and an obligatory interplay between enzymes involved in proteolysis and glycosylation/deglycosylation located in both compartments.     

拟南芥色氨酸与吲哚乙酸生物合成的研究进展

拟南芥色氨酸生物合成途径的研究已逐渐成为植物分子生物学家了解植物基因结构和表达调控最主要的模式系统之一。到目前为止,编码拟南芥色氨酸合成途径的七种酶蛋白的基因已经全部被克隆,并进行了不同程度的分子生物学研究。长期以来,色氨酸一直被认为是植物生长素吲哚乙酸（ＩＡＡ）生物合成（从头合成）的前体物,但近年来人们发现生长素合成的非色氨酸途径可能是其在植物中生物合成的主要途径。植物在不同的发育阶段可能采用不同的方式合成ＩＡＡ。     

The role of the lipid matrix in the biosynthesis of dolichyl-linked oligosaccharides

The enzymes in the dolichol pathway are membrane-proteins that utilize a combination of hydrophilic and extremely hydrophobic substrates. The enzymes in this pathway that have been purified and characterized to any extent have either been shown to be stabilized by mixed phospholipid/detergent micelles, or else require a lipid matrix for catalytic activity. Further understanding of the mechanisms of these essential enzymes may require developing methods for the reconstitution of the glycosyltransferases and their hydrophobic substrates in appropriate lipid matrices. Abbreviations: CHO, Chinese hamster ovary; Dol, dolichol; DAG, diacylglycerol; DOPC, dioleolylphosphatidylcholine; DOPE, dioleolyphosphatidylethanolamine; ER, endoplasmic reticulum; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol This revised version was published online in November 2006 with corrections to the Cover Date.     

Ubiquitin-based anticancer therapy: Carpet bombing with proteasome inhibitors vs surgical strikes with E1, E2, E3, or DUB inhibitors

The proteasome inhibitor bortezomib remains the only ubiquitin pathway effector to become a drug (VELCADE?) and has become a successful treatment for hematological malignancies. While producing a global cellular effect, proteasome inhibitors have not triggered the catastrophe articulated initially in terms such as "buildup of cellular garbage". Proteasome inhibitors, in fact, do have a therapeutic window, although in the case of the prototype bortezomib it is small owing to peripheral neuropathy, myelosuppression and, as recently reported, cardiotoxicity [1]. Currently, several second-generation molecules are undergoing clinical evaluation to increase this window. An alternative strategy is to target ubiquitin pathway enzymes acting at non-proteasomal sites-E1, E2, and E3, associated with ubiquitin conjugation, and deubiquitylating enzymes ("DUBs")-that act locally on selected targets rather than on the whole cell. Inhibitors (or activators, in some cases) of these enzymes should be developable as selective antitumor agents with toxicity profiles superior to that of bortezomib. Various therapeutic hypotheses follow from known cellular mechanisms of these target enzymes; most hypotheses relate to cancer, reminiscent of the FDA-approved protein kinase inhibitors now marketed. Since ubiquitin tagging controls the cellular content, activity, or compartmentation of proteins associated with disease, inhibitors or activators of ubiquitin conjugation or deconjugation are predicted to have an impact on disease. For practical and empirical reasons, inhibitors of ubiquitin pathway enzymes have been the favored therapeutic avenue. In approximately the time that has elapsed since the approval of bortezomib in 2003, there has been some progress in developing potential anticancer drugs that target various ubiquitin pathway enzymes. An E1 inhibitor and inhibitors of E3 are now in clinical trial, with some objective responses reported. Appropriate assays and/or rational design may uncover improved inhibitors of these enzymes, as well as E2 and DUBs, for further development. Presently, it should become clear whether one or both of the two general strategies for ubiquitin-based drug discovery will lead to truly superior new medicines for cancer and other diseases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Deoxyxylulose phosphate pathway to terpenoids

Recently, a mevalonate-independent pathway was discovered in bacteria and plants that leads to the formation of isopentenyl diphosphate and dimethylallyl diphosphate, the two basic precursors of isoprenoids. Although many details of the widely distributed pathway are unknown, some intermediates, mechanisms, enzymes and genes of this novel route have been identified. Information on this pathway could provide the basis for the development of new antibiotics, herbicides and antimalarials.     

Modeling catalytic reaction mechanisms in glycoside hydrolases

Modeling catalysis in carbohydrate-active enzymes is a daunting challenge because of the high flexibility and diversity of both enzymes and carbohydrates. Glycoside hydrolases (GHs) are an illustrative example, where conformational changes and subtle interactions have been shown to be critical for catalysis. GHs have pivotal roles in industry (e.g. biofuel or detergent production) and biomedicine (e.g. targets for cancer and diabetes), and thus, a huge effort is devoted to unveil their molecular mechanisms. Besides experimental techniques, computational methods have served to provide an in-depth understanding of GH mechanisms, capturing complex reaction coordinates and the conformational itineraries that substrates follow during the whole catalytic pathway, providing a framework that ultimately may assist the engineering of these enzymes and the design of new inhibitors.     

Molecular biology of the C3 photosynthetic carbon reduction cycle

In recent years the enzymes of the C₃ photosynthetic carbon reduction (PCR) cycle have been studied using the techniques of molecular biology. In this review we discuss the primary protein sequences and structural predictions that have been made for a number of these enzymes, which, with the input of crystallographic analysis, gives the opportunity to understand the mechanisms of enzyme activity.The genome organisation and gene structure of the PCR enzymes is another area which has recently expanded, and we discuss the regulation of the genes encoding these enzymes and the complex interaction of various factors which influence their expression.