Beskrivelse
Metabolon formation and metabolic channelling enable plants and other organisms to optimize product formation, avoid undesired metabolic interference and to rapidly respond to environmental challenges. Formation of metabolons including e.g. glycosyltransferases and methyltransferases may control the promiscuity of the individual members of these multienzyme families and enable the plant to respond in a coordinated and swift manner to developmentally required re-organization of metabolism or to environmental challenges such as insect or pathogen attack. The deployment of defence reactions may also be restricted to a specific site within the cell and orchestrated by the microtubules and by multimerization of the enzymes involved. New experimental approaches within structural biology provide evidence for the general occurrence of these assemblies of co-operating enzymes also within the synthesis of secondary metabolites including polyphenols with special focus on the flavonoids and iso-flavonoids. At the cellular level, channeling of substrates to their target enzymes is facilitated by the compartmentation of the cell into different organelles and sub-structures thereof. This serves to co-localize and optimize the concentrations of enzymes and their substrates. At the molecular level, yet another increase in substrate concentration is gained by the formation of metabolons (i.e. multienzyme complexes). Metabolon formation serves several purposes: (1) to improve catalytic efficiency by channeling an intermediate that is formed at one active site of an enzyme to the active site of the next enzyme; (2) to relieve kinetic constraints that result from the dilution of intermediates into the bulk phase of the cell; (3) to secure swift conversion of labile and/or toxic intermediates into more stable and less toxic constituents by sequestration; (4) to prevent compounds that might exert an inhibitory effect on the enzyme from reaching the active site; (5) to control and co-ordinate metabolic cross-talk by intermediates shared between different metabolic pathways; (6) to orchestrate swift re-direction of metabolism by the formation of new metabolons that have altered polypeptide composition and product out-put [1,2]. The advantages associated with the organization of a portion of or of an entire biosynthetic pathway in a metabolon are thus many-fold. Metabolon formation typically involves specific interactions between several ‘soluble’ enzymes that might be anchored to a membrane either by membrane-bound structural proteins that serve as ‘nucleation’ sites for metabolon formation or by membrane-bound proteins, such as cytochrome P450s (CYPs), that directly catalyze one or more of the sequential channeled reactions. Mounting evidence indicates that even pathways that were once thought to encompass only soluble enzymes are subject to subcellular structuring that involves metabolon formation [3]. The focus of this review is metabolon formation and metabolic channelling in the synthesis of plant phenolics.. Plants produce an immense number of secondary metabolites, most of which have diverse and highly complex structures. A limited number of key genes encode the enzymes that are responsible for the synthesis of the pivotal backbone structures that constitute the hallmarks of the different classes of natural products including polyphenols and great progress has been made in the identification of these genes. The subsequent decoration of the backbone structures generates the huge diversity of plant secondary products. The large majority of these decoration processes are mediated by a limited number of enzyme classes, such as oxygenases, glycosyl-, methyl- and acyltransferases, which are all encoded by multigene families. Many of these downstream enzymes are regioselective or regiospecific rather than highly substrate specific. The positioning of enzymes that have broad substrate specificity downstream of the conserved early pivotal enzymes of plant secondary metabolism opens the possibility of producing new secondary compounds without major re-structuring of the enzyme complement of the cell. Metabolic channeling and metabolon formation provide the key to resolving and avoiding potential negative interference in plant natural product formation by narrowing substrate specificity of promiscuous enzymes as a result of conformational changes upon binding or because binding into the metabolon prevents access of unwanted substrates. A single glycosyl-, methyl- or acyltransferase might possess the ability to bind to different metabolons. In this manner, the possibility of combinatorially defined substrate specificity might explain how the desired substrate specificity is achieved with a minimum number of enzymes [4]. New experimental approaches within structural biology including small-angle X-ray scattering, single-particle cryo-electron microscopy, mass spectrometry of complexes, co-localization studies using anti-bodies or fluorescent labelled proteins, NMR based flux analyses, proteomics and yeast two hybrid assays have provided a lot more knowledge about the role of metabolons in cell metabolism [2,5]. A large number of plant polyphenols are derived from the shikimate pathway. The structure of the dehydroquinate dehydratase-shikimate dehydrogenase enzyme system in Arabidopsis has been solved. The two separate polypeptides are organized to form a holoenzyme in which the two active sites are facing each other. Clearly, this would be envisioned to facilitate intermediate transfer [2]. Pathways in which evidence for metabolon formation has been obtained include cyanogenic glucosides, flavonoids and iso-flavonoids. Polyphennols do not necessarily represent metabolic end products. Evidence demonstrates that when not in demand, the phenolic cyanogenic glucoside dhurrin may be subjected to endogenous turn-over involving no release of hydrogen cyanide. The process is dependent on formation of nitrilase heteromers since the homomers do not possess this activity. This illustrates how plants may take advantage of combinatorial biochemistry to acquire new specific catalytic properties using the arsenal of enzymes already present [6]. Metabolon formation most likely plays a great importance in the activation of polyphenolic phytoanticipins by b-glucosidases. This is manifested by multimerization of the b-glucosidase required for activation of avenoacosides in oat. The multimeric assembly of the b-glucosidase involves linear stacking of hollow trimeric units resulting in the formation of an extended fibril with a hollow tunnel exposing the active sites and improving enzyme kinetics [7]. Understanding the molecular sociology of the cell i.e. how the proteins present in a cell interact and are spatially arranged within functional modules is imperative to understand cell function and to devise feasible routes for predicted metabolic engineering of plant phenolics by design of effective molecular machines in plants. The heterogeneity and dynamics of these transient functional assemblies poses a big challenge for future research.| Periode | 10 jul. 2008 |
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| Begivenhedstitel | XXIVth International Conference on Polyphenols. |
| Begivenhedstype | Konference |
| Placering | Salamanca, SpanienVis på kort |