Multienzyme Assemblies Research Articles

Cascade biocatalysis by multienzyme-nanoparticle assemblies.

Multienzyme complexes are of paramount importance in biosynthesis in cells. Yet, how sequential enzymes of cascade catalytic reactions synergize their activities through spatial organization remains elusive. Recent development of site-specific protein-nanoparticle conjugation techniques enables us to construct multienzyme assemblies using nanoparticles as the template. Sequential enzymes in menaquinone biosynthetic pathway were conjugated to CdSe-ZnS quantum dots (QDs, a nanosized particulate material) through metal-affinity driven self-assembly. The assemblies were characterized by electrophoretic methods, the catalytic activities were monitored by reverse-phase chromatography, and the composition of the multienzyme-QD assemblies was optimized through a progressive approach to achieve highly efficient catalytic conversion. Shorter enzyme-enzyme distance was discovered to facilitate intermediate transfer, and a fine control on the stoichiometric ratio of the assembly was found to be critical for the maximal synergy between the enzymes. Multienzyme-QD assemblies thereby provide an effective model to scrutinize the synergy of cascade enzymes in multienzyme complexes.

Type I pyridoxal 5′-phosphate dependent enzymatic domains embedded within multimodular nonribosomal peptide synthetase and polyketide synthase assembly lines

BackgroundPyridoxal 5′-phosphate (PLP)-dependent enzymes of fold type I, the most studied structural class of the PLP-dependent enzyme superfamily, are known to exist as stand-alone homodimers or homotetramers. These enzymes have been found also embedded in multimodular and multidomain assembly lines involved in the biosynthesis of polyketides (PKS) and nonribosomal peptides (NRPS). The aim of this work is to provide a proteome-wide view of the distribution and characteristics of type I domains covalently integrated in these assemblies in prokaryotes.ResultsAn ad-hoc Hidden Markov profile was calculated using a sequence alignment derived from a multiple structural superposition of distantly related PLP-enzymes of fold type I. The profile was utilized to scan the sequence databank and to collect the proteins containing at least one type I domain linked to a component of an assembly line in bacterial genomes. The domains adjacent to a carrier protein were further investigated. Phylogenetic analysis suggested the presence of four PLP-dependent families: Aminotran_3, Beta_elim_lyase and Pyridoxal_deC, occurring mainly within mixed NRPS/PKS clusters, and Aminotran_1_2 found mainly in PKS clusters. Sequence similarity to the reference PLP enzymes with solved structures ranged from 24 to 42% identity. Homology models were built for each representative type I domain and molecular docking simulations with putative substrates were carried out. Prediction of the protein-protein interaction sites evidenced that the surface regions of the type I domains embedded within multienzyme assemblies were different from those of the self-standing enzymes; these structural features appear to be required for productive interactions with the adjacent domains in a multidomain context.ConclusionsThis work provides a systematic view of the occurrence of type I domain within NRPS and PKS assembly lines and it predicts their structural characteristics using computational methods. Comparison with the corresponding stand-alone enzymes highlighted the common and different traits related to various aspects of their structure-function relationship. Therefore, the results of this work, on one hand contribute to the understanding of the functional and structural diversity of the PLP-dependent type I enzymes and, on the other, pave the way to further studies aimed at their applications in combinatorial biosynthesis.

‘Gestalt,’ Composition and Function of the Trypanosoma brucei Editosome

RNA editing describes a chemically diverse set of biomolecular reactions in which the nucleotide sequence of RNA molecules is altered. Editing reactions have been identified in many organisms and frequently contribute to the maturation of organellar transcripts. A special editing reaction has evolved within the mitochondria of the kinetoplastid protozoa. The process is characterized by the insertion and deletion of uridine nucleotides into otherwise nontranslatable messenger RNAs. Kinetoplastid RNA editing involves an exclusive class of small, noncoding RNAs known as guide RNAs. Furthermore, a unique molecular machinery, the editosome, catalyzes the process. Editosomes are megadalton multienzyme assemblies that provide a catalytic surface for the individual steps of the reaction cycle. Here I review the current mechanistic understanding and molecular inventory of kinetoplastid RNA editing and the editosome machinery. Special emphasis is placed on the molecular morphology of the editing complex in order to correlate structural features with functional characteristics.

Multimeric Options for the Auto-Activation of the Saccharomyces cerevisiae FAS Type I Megasynthase

The fungal type I fatty acid synthase (FAS) is a 2.6 MDa multienzyme complex, catalyzing all necessary steps for the synthesis of long acyl chains. To be catalytically competent, the FAS must be activated by a posttranslational modification of the central acyl carrier domain (ACP) by an intrinsic phosphopantetheine transferase (PPT). However, recent X-ray structures of the fungal FAS revealed a barrel-shaped architecture, with PPT located at the outside of the barrel wall, spatially separated from the ACP caged in the inner volume. This separation indicated that the activation has to proceed before the assembly to the mature complex, in a conformation where the ACP and PPT domains can meet. To gain insight into the auto-activation reaction and also into the fungal FAS assembly pathway, we structurally and functionally characterized the Saccharomyces cerevisiae FAS type I PPT as part of the multienzyme protein and as an isolated domain.

Three-Dimensional Structure of the Respiratory Chain Supercomplex I1III2IV1 from Bovine Heart Mitochondria,

The respiratory chain complexes can arrange into multienzyme assemblies, so-called supercomplexes. We present the first 3D map of a respiratory chain supercomplex. It was determined by random conical tilt electron microscopy analysis of a bovine supercomplex consisting of complex I, dimeric complex III, and complex IV (I1III2IV1). Within this 3D map the positions and orientations of all the individual complexes in the supercomplex were determined unambiguously. Furthermore, the ubiquinone and cytochrome c binding sites of each complex in the supercomplex could be located. The mobile electron carrier binding site of each complex was found to be in proximity to the binding site of the succeeding complex in the respiratory chain. This provides structural evidence for direct substrate channeling in the supercomplex assembly with short diffusion distances for the mobile electron carriers.

2-Oxo acid dehydrogenase complexes of multicellular organisms.

Investigation of the 2-oxo acid dehydrogenase multienzyme complexes (Mr ≈ 106−107 Da) has many facets, from studies of organic catalysis within the protein structure to unraveling general principles of multienzyme complex assembly [[1-5]]. It bridges bioorganic chemistry and cell biology, and is both fascinating in itself and important methodologically. In particular, 2-oxo acid dehydrogenase complexes are inexhaustible source of valuable data for understanding the biological significance of protein assemblies. A new type of symmetry-driven dynamics of the complexes has also been suggested [[6]]: the ‘breathing’ of the macromolecular structure is presumed to have an impact on its catalysis and regulation, although the exact relationship between the dynamics and function is still to be established. Studies of the transfer of intermediates within the complexes have revealed a multiple random coupling mechanism provided by a network of covalently attached lipoyl groups in the complex multimeric core, with catalysis accelerated in part by mechanical movements of domains [[7]]. In this Minireview series, we demonstrate the benefits of multienzyme assemblies for integrated functional regulation. Thus, only one molecule of the pyruvate dehydrogenase kinase may be employed by the 60-meric core to rapidly inactivate all molecules of the first component enzyme in a process sensitive to the chemical state of the lipoyl groups in the assembly (Minireview in this series, Roche et al.). Another advantage of an extended complex is its ability to create compartments of biologically active intermediates. This not only increases the local concentration of the intermediate, but also influences its reactivity, enabling reactions that are not efficient with the same number of separate molecules (Minireviews in this series, Bunik and Roche et al.). What allows the enzyme complexes to satisfy the additional requirements of multicellular organisms? We address this question by focusing on the regulation of the 2-oxo acid dehydrogenase complexes from higher plants and animals. The first Minireview presents data on specific intra-organellar regulation of mammalian complexes by mitochondrial thioredoxin. Such regulation appears to have evolved along with protein compartmentalization within the mitochondria. The second Minireview considers peculiarities of the 2-oxo acid dehydrogenase complexes within the different cellular compartments, mitochondria and chloroplasts. Specific metabolic requirements of the organelles are met through modified regulation of the same catalytic process. This principle is also applied at the tissue level. Study of the pyruvate dehydrogenase regulation by phosphorylation (third Minireview) demonstrates that the enzyme function is adjusted to metabolic demands of different tissues by the tissue-specific kinases and phosphatases. Thus, multicellular systems are characterized by an increased complexity of the enzyme regulation; organization of proteins into multienzyme structures is a powerful means to extend the opportunities for regulation. Victoria Bunik is currently a senior scientist of the A.N. Belozersky Institute of Physico-Chemical Biology, Moscow M.V. Lomonosov State University looking at the structure–function relationship in the 2-oxo acid dehydrogenase complexes following her PhD (1987) and Doctor of Chemical Sciences (2001) in the same field. In 1992 she was awarded the Moscow M.V. Lomonosov State University award for the best research among young scientists. Victoria has collaborated with several laboratories in Europe and the USA, including a period (1996–1997) of research at Tuebingen University in Germany provided for by a fellowship from the Alexander von Humboldt Foundation (Bonn, Germany), which she received in 1995.

Domain Analysis of the Molecular Recognition Features of Aromatic Polyketide Synthase Subunits

Bacterial aromatic polyketide synthases (PKSs) are a family of homologous multienzyme assemblies that catalyze the biosynthesis of numerous polyfunctional aromatic natural products. In the absence of direct insights into their structures, the use of gene fusions can be a powerful tool for understanding the structural basis for their properties. A series of truncated and hybrid proteins were constructed and analyzed within a family of PKS subunits, designated aromatases/cyclases (ARO/CYCs). When expressed alone, neither the N-terminal nor the C-terminal domain of the actinorhodin (act) or the griseusin (gris) ARO/CYC exhibited substantial aromatase activity. However, in the presence of each other, the half proteins were active. Furthermore, analysis of a set of hybrid proteins derived from the act and gris ARO/CYCs allowed us to localize the chain length dependence of this aromatase activity to their N-terminal domains. Unexpectedly, however, when the C-terminal domain of the gris ARO/CYC was expressed in a context where aromatase activity was absent, it could modulate the chain length specificity of the tetracenomycin (tcm) minimal PKS, leading to the formation of a novel 18-carbon product in addition to the expected 20-carbon one. It was also found that monodomain ARO/CYCs such as tcmN cannot substitute for the the N-terminal domain of didomain ARO/CYCs, even though they exhibit high sequence similarity with the N-terminal domain. Together, these results illustrate the utility of protein engineering approaches for dissecting the structure-function relationships of PKS subunits and for the generation of mutant alleles with novel biosynthetic properties.

Evidence for two catalytically independent clusters of active sites in a functional modular polyketide synthase.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multifunctional enzymes are organized into "modules", where each module contains active sites homologous to those of higher eucaryotic fatty acid synthases (FASs). Like FASs, modular PKSs are known to be dimers. Here we provide functional evidence for the existence of two catalytically independent clusters of active sites within a modular PKS. In three bimodular derivatives of DEBS, the ketosynthase domain of module 1 (KS-1) or module 2 (KS-2) or the acyl carrier protein domain of module 2 (ACP-2) was inactivated via site-directed mutagenesis. As expected, the purified proteins were unable to catalyze polyketide synthesis (although the KS-1 mutant could convert a diketide thioester into the predicted triketide lactone). Remarkably however, the KS-1/KS-2 and the KS-2/ACP-2 mutant pairs could efficiently complement each other and catalyze polyketide formation. In contrast, the KS-1 and ACP-2 mutants did not complement each other. On the basis of these and other results, a model is proposed in which the individual modules of a PKS dimer form head-to-tail homodimers, thereby generating two equivalent and independent clusters of active sites for polyketide biosynthesis. Specifically, each subunit contributes half of the KS and ACP domains in each cluster. A similar complementation approach should also be useful in dissecting the organization of the remaining types of active sites within this family of multienzyme assemblies. Finally, blocked systems, such as the KS-1 mutant described here, present a new strategy for the noncompetitive conversion of unnatural substrates into polyketides by modular PKSs.

Ultrastructural alteration of the mitochondrial electron transport chain involving electron leak possible basis of “respiratory impairment” in certain tumors

It is proposed that the lower respiratory rates found in certain tumor mitochondria as compared to that in corresponding non-neoplastic tissue results initially from the considerable alteration or deletion of the mechanochemical effector system which is responsible for the swelling and contraction of mitochondria. The effector system, which is membranelocalized, is linked to the respiratory chain in a position adjacent to the coupling sequence which is responsible for phosphorylation. The considerable loss or disappearance of both the “high amplitude” swelling and contraction functions in tumor mitochondria indicate severe alteration or deletion of the effector system. Since it is well-established that the respiratory multienzyme assemblies in the membrane are lipoid coated (for the insulation of electron transport from the aqueous phase), the alteration or deletion of the effector system cannot fail to bring about a break-up of the lipoid coating; this break-up is possibly compounded by the well-known alterations of fatty acid, phospholipid and cholesterol metabolism in tumors. The working hypothesis is developed that, because of electron leak through this lesion(s), a portion of the electron flux resulting from substrate dehydrogenation does not reach the terminal acceptor, oxygen, although the rate of dehydrogenation may remain unaltered; hence, decrease of the respiratory rate. Corollaries to this situation are analyzed.