ABSTRACT
Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognize ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerization, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, and assemble into a high molecular weight oligomeric complex, the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å2 and a smaller one of 415 Å2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerization of DTX3L is important for the DTX3L-PARP9 complex to read mono-ADP-ribosylation on a ligand-regulated transcription factor.
Subject(s)
Reading , Receptors, Androgen , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Adenosine Diphosphate Ribose/metabolismABSTRACT
Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognise ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerisation, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, they assemble into a high molecular weight oligomeric complex, but the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å2 and a smaller one of 415 Å2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerisation of DTX3L is important for mono-ADP-ribosylation reading by the DTX3L-PARP9 complex and to a ligand-regulated transcription factor.
ABSTRACT
ADP-ribosylation is a post-translational modification catalyzed by writer enzymes - ADP-ribosyltransferases. The modification is part of many signaling events, can modulate the function and stability of target proteins, and often results in the recruitment of reader proteins that bind to the ADP-ribosyl groups. Erasers are integral actors in these signaling events and reverse the modification. ADP-ribosylation can be targeted with therapeutics and many inhibitors against writers exist, with some being in clinical use. Inhibitors against readers and erasers are sparser and development of these has gained momentum only in recent years. Drug discovery has been hampered by the lack of specific tools, however many significant advances in the methods have recently been reported. We discuss assays used in the field with a focus on methods allowing efficient identification of small molecule inhibitors and profiling against enzyme families. While human proteins are focused, the methods can be also applied to bacterial toxins and virus encoded erasers that can be targeted to treat infectious diseases in the future.
Subject(s)
ADP Ribose Transferases , Protein Processing, Post-Translational , ADP Ribose Transferases/metabolism , Adenosine Diphosphate , Biological Assay , Drug Discovery , HumansABSTRACT
Sialic acid residues found as terminal monosaccharides in various types of glycan chains in cell surface glycoproteins and glycolipids have been identified as important contributors of cell-cell interactions in normal vs. abnormal cellular behavior and are pivotal in diseases such as cancers. In vertebrates, sialic acids are attached to glycan chains by a conserved subset of sialyltransferases with different enzymatic and substrate specificities. ST6Gal I is a sialyltransferase using activated CMP-sialic acids as donor substrates to catalyze the formation of a α2,6-glycosidic bond between the sialic acid residue and the acceptor disaccharide LacNAc. Understanding sialyltransferases at the molecular and structural level shed light into their function. We present here two human ST6Gal I structures, which show for the first time the enzyme in the unliganded state and with the full donor substrate CMP-Neu5Ac bound. Comparison of these structures reveal flexibility of the catalytic loop, since in the unliganded structure Tyr354 adopts a conformation seen also as an alternate conformation in the substrate bound structure. CMP-Neu5Ac is bound with the side chain at C5 of the sugar residue directed outwards at the surface of the protein. Furthermore, the exact binding mode of the sialic acid moiety of the substrate directly involves sialylmotifs L, S and III and positions the sialylmotif VS in the immediate vicinity. We also present a model for the ternary complex of ST6Gal I with both the donor and the acceptor substrates.
Subject(s)
Antigens, CD/chemistry , Cytidine Monophosphate/analogs & derivatives , Cytidine Monophosphate/chemistry , Sialic Acids/chemistry , Sialyltransferases/chemistry , Animals , Humans , Monosaccharides/chemistry , Polysaccharides/chemistry , Substrate Specificity/physiology , beta-D-Galactoside alpha 2-6-SialyltransferaseABSTRACT
ß-1,4-Galactosyltransferase 1 (B4GALT1) and ST6 ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) catalyze the successive addition of terminal ß-1,4-linked galactose and α-2,6-linked sialic acid to N-glycans. Their exclusive interaction in the Golgi compartment is a prerequisite for their full catalytic activity, whereas a lack of this interaction is associated with cancers and hypoxia. To date, no structural information exists that shows how glycosyltransferases functionally assemble with each other. Using molecular docking simulations to predict interaction surfaces, along with mutagenesis screens and high-throughput FRET analyses in live cells to validate these predictions, we show here that B4GALT1 and ST6GAL1 interact via highly charged noncatalytic surfaces, leaving the active sites exposed and accessible for donor and acceptor substrate binding. Moreover, we found that the assembly of ST6GAL1 homomers in the endoplasmic reticulum before ST6GAL1 activation in the Golgi utilizes the same noncatalytic surface, whereas B4GALT1 uses its active-site surface for assembly, which silences its catalytic activity. Last, we show that the homomeric and heteromeric B4GALT1/ST6GAL1 complexes can assemble laterally in the Golgi membranes without forming cross-cisternal contacts between enzyme molecules residing in the opposite membranes of each Golgi cisterna. Our results provide detailed mechanistic insights into the regulation of glycosyltransferase interactions, the transitions between B4GALT1 and ST6GAL1 homo- and heteromers in the Golgi, and cooperative B4GALT1/ST6GAL1 function in N-glycan synthesis.
Subject(s)
Antigens, CD/chemistry , Galactosyltransferases/chemistry , Molecular Docking Simulation , Protein Multimerization , Sialyltransferases/chemistry , Animals , Antigens, CD/metabolism , Binding Sites , CHO Cells , COS Cells , Chlorocebus aethiops , Cricetinae , Cricetulus , Fluorescence Resonance Energy Transfer , Galactosyltransferases/metabolism , Golgi Apparatus/metabolism , Sialyltransferases/metabolism , Static ElectricityABSTRACT
Glycosylation, a common modification of cellular proteins and lipids, is often altered in diseases and pathophysiological states such as hypoxia, yet the underlying molecular causes remain poorly understood. By utilizing lectin microarray glycan profiling, Golgi pH and redox screens, we show here that hypoxia inhibits terminal sialylation of N- and O-linked glycans in a HIF- independent manner by lowering Golgi oxidative potential. This redox state change was accompanied by loss of two surface-exposed disulfide bonds in the catalytic domain of the α-2,6-sialyltransferase (ST6Gal-I) and its ability to functionally interact with B4GalT-I, an enzyme adding the preceding galactose to complex N-glycans. Mutagenesis of selected cysteine residues in ST6Gal-I mimicked these effects, and also rendered the enzyme inactive. Cells expressing the inactive mutant, but not those expressing the wild type ST6Gal-I, were able to proliferate and migrate normally, supporting the view that inactivation of the ST6Gal-I help cells to adapt to hypoxic environment. Structure comparisons revealed similar disulfide bonds also in ST3Gal-I, suggesting that this O-glycan and glycolipid modifying sialyltransferase is also sensitive to hypoxia and thereby contribute to attenuated sialylation of O-linked glycans in hypoxic cells. Collectively, these findings unveil a previously unknown redox switch in the Golgi apparatus that is responsible for the catalytic activation and cooperative functioning of ST6Gal-I with B4GalT-I.
Subject(s)
Galactosyltransferases/metabolism , Golgi Apparatus/metabolism , Oxidation-Reduction , Sialyltransferases/metabolism , Animals , Catalysis , Cell Line , Cell Movement , Cell Proliferation , Disulfides/metabolism , Galactosyltransferases/chemistry , Humans , Hydrogen-Ion Concentration , Hypoxia-Inducible Factor 1/genetics , Hypoxia-Inducible Factor 1/metabolism , Models, Molecular , Molecular Conformation , Polysaccharides/metabolism , Sialyltransferases/chemistry , beta-D-Galactoside alpha 2-6-SialyltransferaseABSTRACT
Branching and processing of N-glycans in the medial-Golgi rely both on the transport of the donor UDP-N-acetylglucosamine (UDP-GlcNAc) to the Golgi lumen by the SLC35A3 nucleotide sugar transporter (NST) as well as on the addition of the GlcNAc residue to terminal mannoses in nascent N-glycans by several linkage-specific N-acetyl-glucosaminyltransferases (MGAT1-MGAT5). Previous data indicate that the MGATs and NSTs both form higher order assemblies in the Golgi membranes. Here, we investigate their specific and mutual interactions using high-throughput FRET- and BiFC-based interaction screens. We show that MGAT1, MGAT2, MGAT3, MGAT4B (but not MGAT5) and Golgi alpha-mannosidase IIX (MAN2A2) form several distinct molecular assemblies with each other and that the MAN2A2 acts as a central hub for the interactions. Similar assemblies were also detected between the NSTs SLC35A2, SLC35A3, and SLC35A4. Using in vivo BiFC-based FRET interaction screens, we also identified novel ternary complexes between the MGATs themselves or between the MGATs and the NSTs. These findings suggest that the MGATs and the NSTs self-assemble into multi-enzyme/multi-transporter complexes in the Golgi membranes in vivo to facilitate efficient synthesis of complex N-glycans.
Subject(s)
Golgi Apparatus/metabolism , Monosaccharide Transport Proteins/metabolism , Multienzyme Complexes/metabolism , N-Acetylglucosaminyltransferases/metabolism , Nucleotide Transport Proteins/metabolism , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Polysaccharides/metabolism , Uridine Diphosphate N-Acetylglucosamine/metabolism , alpha-Mannosidase/metabolismABSTRACT
17ß-Hydroxysteroid dehydrogenases (HSD17B) catalyze the oxidation/reduction of 17ß-hydroxy/keto group in position C17 in C18- and C19 steroids. Most HSD17Bs are also catalytically active with substrates other than steroids. A subset of these enzymes is able to process thioesters of carboxylic acids. This group of enzymes includes HSD17B4, HSD17B8, HSD17B10 and HSD17B12, which execute reactions in intermediary metabolism, participating in peroxisomal ß-oxidation of fatty acids, mitochondrial oxidation of 3R-hydroxyacyl-groups, breakdown of isoleucine and fatty acid chain elongation in endoplasmic reticulum. Divergent substrate acceptance capabilities exemplify acquirement of catalytic site adaptiveness during evolution. As an additional common feature these HSD17Bs are multifunctional enzymes that arose either via gene fusions (HSD17B4) or are incorporated as subunits into multifunctional protein complexes (HSD17B8 and HSD17B10). Crystal structures of HSD17B4, HSD17B8 and HSD17B10 give insight into their structure-function relationships. Thus far, deficiencies of HSD17B4 and HSD17B10 have been assigned to inborn errors in humans, underlining their significance as enzymes of metabolism.
Subject(s)
17-Hydroxysteroid Dehydrogenases/metabolism , Esters/metabolism , 17-Hydroxysteroid Dehydrogenases/chemistry , Animals , Disease , Fatty Acids, Unsaturated/metabolism , Humans , Mitochondria/metabolism , RNA/metabolismABSTRACT
AIMS: Carcinoembryonic antigen (CEACAM5, CEA) is a known tumor marker for colorectal cancer that localizes in a polarized manner to the apical surface in normal colon epithelial cells whereas in cancer cells it is present at both the apical and basolateral surfaces of the cells. Since the Golgi apparatus sorts and transports most proteins to these cell surface domains, we set out here to investigate whether any of the factors commonly associated with tumorigenesis, including hypoxia, generation of reactive oxygen species (ROS), altered redox homeostasis, or an altered Golgi pH, are responsible for mistargeting of CEA to the basolateral surface in cancer cells. RESULTS: Using polarized nontumorigenic Madin-Darby canine kidney (MDCK) cells and CaCo-2 colorectal cancer cells as targets, we show that apical delivery of CEA is not affected by hypoxia, ROS, nor changes in the Golgi redox state. Instead, we find that an elevated Golgi pH induces basolateral targeting of CEA and increases its TX-100 solubility, indicating impaired association of CEA with lipid rafts. Moreover, disruption of lipid rafts by methyl-ß-cyclodextrin induced accumulation of the CEA protein at the basolateral surface in MDCK cells. Experiments with the glycosylphosphatidylinositol (GPI)-anchorless CEA mutant and CEA-specific GPI-anchored enhanced green fluorescent protein (EGFP-GPI) fusion protein revealed that the GPI-anchor was critical for the pH-dependent apical delivery of the CEA in MDCK cells. Innovation and Conclusion: The findings indicate that an abnormal Golgi pH homeostasis in cancer cells is an important factor that causes mistargeting of CEA to the basolateral surface of cancer cells via inhibiting its GPI-anchor-mediated association with lipid rafts.
Subject(s)
Carcinoembryonic Antigen/metabolism , Glycosylphosphatidylinositols/metabolism , Golgi Apparatus/metabolism , Homeostasis , Membrane Microdomains/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Animals , Caco-2 Cells , Dogs , Humans , Hydrogen-Ion Concentration , Madin Darby Canine Kidney Cells/metabolismABSTRACT
Most glycosyltransferases, including B4GalT1 (EC 2.4.1.38), are known to assemble into enzyme homomers and functionally relevant heteromers in vivo. However, it remains unclear why and how these enzymes interact at the molecular/atomic level. Here, we solved the crystal structure of the wild-type human B4GalT1 homodimer. We also show that B4GalT1 exists in a dynamic equilibrium between monomer and dimer, since a purified monomer reappears as a mixture of both and as we obtained crystal forms of the monomer and dimer assemblies in the same crystallization conditions. These two crystal forms revealed the unliganded B4GalT1 in both the open and the closed conformation of the Trp loop and the lid regions, responsible for donor and acceptor substrate binding, respectively. The present structures also show the lid region in full in an open conformation, as well as a new conformation for the GlcNAc acceptor loop (residues 272-288). The physiological relevance of the homodimer in the crystal was validated by targeted mutagenesis studies coupled with FRET assays. These showed that changing key catalytic amino acids impaired homomer formation in vivo. The wild-type human B4GalT1 structure also explains why the variant proteins used for crystallization in earlier studies failed to reveal the homodimers described in this study.
Subject(s)
Galactosyltransferases/metabolism , Animals , COS Cells , Chlorocebus aethiops , Escherichia coli , Galactosyltransferases/chemistry , Galactosyltransferases/genetics , Galactosyltransferases/isolation & purification , Humans , Models, Molecular , Mutation , Protein Conformation , Protein Domains , Protein MultimerizationABSTRACT
Glycosyltransferases (GTases) transfer sugar moieties to proteins, lipids or existing glycan or polysaccharide molecules. GTases form an important group of enzymes in the Golgi, where the synthesis and modification of glycoproteins and glycolipids take place. Golgi GTases are almost invariably type II integral membrane proteins, with the C-terminal globular catalytic domain residing in the Golgi lumen. The enzymes themselves are divided into 103 families based on their sequence homology. There is an abundance of published crystal structures of GTase catalytic domains deposited in the Protein Data Bank (PDB). All of these represent either of the two main characteristic structural folds, GT-A or GT-B, or present a variation thereof. Since GTases can function as homomeric or heteromeric complexes in vivo, we have summarized the structural features of the dimerization interfaces in crystal structures of GTases, as well as considered the biochemical data available for these enzymes. For this review, we have considered all 898 GTase crystal structures in the Protein Data Bank and highlight the dimer formation characteristics of various GTases based on 24 selected structures.
Subject(s)
Eukaryota/enzymology , Glycosyltransferases/chemistry , Glycosyltransferases/metabolism , Protein Multimerization , Animals , Crystallography, X-Ray , Databases, Protein , Eukaryotic Cells/enzymology , Eukaryotic Cells/metabolism , Humans , Models, MolecularABSTRACT
Glycosylation is the most common and complex cellular modification of proteins and lipids. It is critical for multicellular life and its abrogation often leads to a devastating disease. Yet, the underlying mechanistic details of glycosylation in both health and disease remain unclear. Partly, this is due to the complexity and dynamicity of glycan modifications, and the fact that not all the players are taken into account. Since late 1960s, a vast number of studies have demonstrated that glycosyltransferases typically form homomeric and heteromeric complexes with each other in yeast, plant and animal cells. To propagate their acceptance, we will summarize here accumulated data for their prevalence and potential functional importance for glycosylation focusing mainly on their mutual interactions, the protein domains mediating these interactions, and enzymatic activity changes that occur upon complex formation. Finally, we will highlight the few existing 3D structures of these enzyme complexes to pinpoint their individual nature and to emphasize that their lack is the main obstacle for more detailed understanding of how these enzyme complexes interact and function in a eukaryotic cell.
Subject(s)
Glycolipids/metabolism , Glycosyltransferases/metabolism , Polysaccharides/metabolism , Proteoglycans/metabolism , Animals , Carbohydrate Sequence , Glycolipids/chemistry , Glycosylation , Glycosyltransferases/chemistry , Golgi Apparatus/chemistry , Golgi Apparatus/metabolism , Humans , Molecular Sequence Data , Polysaccharides/chemistry , Protein Conformation , Protein Interaction Maps , Proteoglycans/chemistryABSTRACT
Molecular basis of D-bifunctional protein (D-BP) deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2) protein. Complementation analysis in vivo in yeast and in vitro enzyme kinetic and stability determinants as well as in silico stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K(m), V(max) and k(cat)) of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.
Subject(s)
17-Hydroxysteroid Dehydrogenases/chemistry , 17-Hydroxysteroid Dehydrogenases/deficiency , Gonadal Dysgenesis, 46,XX/genetics , Hearing Loss, Sensorineural/genetics , Hydro-Lyases/chemistry , Hydro-Lyases/deficiency , Mutation , Peroxisomes/genetics , 17-Hydroxysteroid Dehydrogenases/genetics , Catalytic Domain , Child , Child, Preschool , Cloning, Molecular , Enzyme Stability , Escherichia coli/genetics , Fatty Acids/metabolism , Female , Genetic Complementation Test , Gonadal Dysgenesis, 46,XX/enzymology , Hearing Loss, Sensorineural/enzymology , Humans , Hydro-Lyases/genetics , Kinetics , Lipid Metabolism , Male , Models, Molecular , Oxidation-Reduction , Peroxisomal Multifunctional Protein-2 , Peroxisomes/enzymology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Saccharomyces cerevisiae/genetics , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Multifunctional enzyme type 2 (MFE-2) forms part of the fatty acid ß-oxidation pathway in peroxisomes. MFE-2s from various species reveal proteins with structurally homologous functional domains assembled in different compilations. Crystal structures of all domain types are known. SAXS data from human, fruit fly and Caenorhabditiselegans MFE-2s and their constituent domains were collected, and both ab initio and rigid body models constructed. Location of the putative substrate binding helper domain SCP-2L (sterol carrier protein 2-like), which is not part of MFE-2 protein in every species and not seen as part of any previous MFE-2 structures, was determined. The obtained models of human and C. elegans MFE-2 lend a direct structural support to the idea of the biological role of SCP-2L.
Subject(s)
17-Hydroxysteroid Dehydrogenases/chemistry , 3-Hydroxyacyl CoA Dehydrogenases/chemistry , Alcohol Oxidoreductases/chemistry , Caenorhabditis elegans Proteins/chemistry , Carbon-Oxygen Lyases/chemistry , Drosophila Proteins/chemistry , Enoyl-CoA Hydratase/chemistry , Hydro-Lyases/chemistry , Models, Molecular , Multienzyme Complexes/chemistry , 17-Hydroxysteroid Dehydrogenases/genetics , 17-Hydroxysteroid Dehydrogenases/metabolism , 3-Hydroxyacyl CoA Dehydrogenases/genetics , 3-Hydroxyacyl CoA Dehydrogenases/metabolism , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , Caenorhabditis elegans/enzymology , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Carbon-Oxygen Lyases/genetics , Carbon-Oxygen Lyases/metabolism , Carrier Proteins/chemistry , Dimerization , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Enoyl-CoA Hydratase/genetics , Enoyl-CoA Hydratase/metabolism , Humans , Hydro-Lyases/genetics , Hydro-Lyases/metabolism , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peroxisomal Multifunctional Protein-2 , Peroxisomes/enzymology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Scattering, Small Angle , Solubility , Species Specificity , Synchrotrons , X-Ray DiffractionABSTRACT
All of the peroxisomal ß-oxidation pathways characterized thus far house at least one MFE (multifunctional enzyme) catalysing two out of four reactions of the spiral. MFE type 2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for Drosophila melanogaster MFE-2 (DmMFE-2), complements the Saccharomyces cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure-function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both the structural features, as well as by the fact that hydration and dehydrogenation activities of MFE-2, if produced as separate enzymes, are equally efficient in catalysis as the full-length MFE-2.
Subject(s)
Alcohol Oxidoreductases/metabolism , Carbon-Oxygen Lyases/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Enoyl-CoA Hydratase/metabolism , Multienzyme Complexes/metabolism , Oxidoreductases/metabolism , Alcohol Oxidoreductases/genetics , Animals , Carbon-Oxygen Lyases/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Enoyl-CoA Hydratase/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Enzymologic , Models, Molecular , Multienzyme Complexes/genetics , Oxidoreductases/genetics , Plasmids , Protein Conformation , Protein Folding , Protein Structure, TertiaryABSTRACT
Peroxisomes play an essential role in a number of important metabolic pathways including beta-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various beta-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal beta-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp(249) to Arg(251)) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp(249) to Arg(251) and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.
Subject(s)
17-Hydroxysteroid Dehydrogenases/analysis , 17-Hydroxysteroid Dehydrogenases/genetics , Enoyl-CoA Hydratase/analysis , Enoyl-CoA Hydratase/genetics , Multienzyme Complexes/analysis , Multienzyme Complexes/genetics , Palmitic Acid/chemistry , Peroxisomes/enzymology , Animals , Binding Sites , Diazomethane/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Liver , Molecular Probe Techniques , Mutation, Missense , Oxidation-Reduction , Peroxisomal Multifunctional Protein-2 , Photoaffinity Labels , RatsABSTRACT
BACKGROUND: Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures. RESULTS: The enzymatic glucose release system together with a well-balanced combination of mineral salts and complex medium additives provided high cell densities, high protein yields and a considerably improved proportion of soluble proteins in harvested cells. The cultivation method consists of three steps: 1) controlled growth by glucose-limited fed-batch to OD600 approximately 10, 2) addition of growth boosters together with an inducer providing efficient protein synthesis within a 3 to 6 hours period, and 3) a slow growth period (16 to 21 hours) during which the recombinant protein is slowly synthesized and folded. Cell densities corresponding to 10 to 15 g l(-1) cell dry weight could be achieved with the developed technique. In comparison to standard cultures in LB, Terrific Broth and mineral salt medium, we typically achieved over 10-fold higher volumetric yields of soluble recombinant proteins. CONCLUSIONS: We have demonstrated that by applying the novel EnBase Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce the amount of time and effort needed for downstream processing or process optimization. We claim that the new cultivation system is widely applicable and, as it is very simple to apply, could widely replace standard shake flask approaches.
Subject(s)
Culture Techniques/methods , Escherichia coli/growth & development , Biomass , Culture Media/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Glucose/metabolism , Oxygen/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Fatty acid degradation in most organisms occurs primarily via the beta-oxidation cycle. In mammals, beta-oxidation occurs in both mitochondria and peroxisomes, whereas plants and most fungi harbor the beta-oxidation cycle only in the peroxisomes. Although several of the enzymes participating in this pathway in both organelles are similar, some distinct physiological roles have been uncovered. Recent advances in the structural elucidation of numerous mammalian and yeast enzymes involved in beta-oxidation have shed light on the basis of the substrate specificity for several of them. Of particular interest is the structural organization and function of the type 1 and 2 multifunctional enzyme (MFE-1 and MFE-2), two enzymes evolutionarily distant yet catalyzing the same overall enzymatic reactions but via opposite stereochemistry. New data on the physiological roles of the various enzymes participating in beta-oxidation have been gathered through the analysis of knockout mutants in plants, yeast and animals, as well as by the use of polyhydroxyalkanoate synthesis from beta-oxidation intermediates as a tool to study carbon flux through the pathway. In plants, both forward and reverse genetics performed on the model plant Arabidopsis thaliana have revealed novel roles for beta-oxidation in the germination process that is independent of the generation of carbohydrates for growth, as well as in embryo and flower development, and the generation of the phytohormone indole-3-acetic acid and the signal molecule jasmonic acid.
Subject(s)
Metabolic Networks and Pathways , Peroxisomes/physiology , Acetyl-CoA C-Acyltransferase/metabolism , Acyl-CoA Oxidase/metabolism , Animals , Arabidopsis/genetics , Arabidopsis/physiology , Multienzyme Complexes/metabolism , Oxidation-Reduction , Substrate SpecificityABSTRACT
(3R)-hydroxyacyl-CoA dehydrogenase is part of multifunctional enzyme type 2 (MFE-2) of peroxisomal fatty acid beta-oxidation. The MFE-2 protein from yeasts contains in the same polypeptide chain two dehydrogenases (A and B), which possess difference in substrate specificity. The crystal structure of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase AB heterodimer, consisting of dehydrogenase A and B, determined at the resolution of 2.2A, shows overall similarity with the prototypic counterpart from rat, but also important differences that explain the substrate specificity differences observed. Docking studies suggest that dehydrogenase A binds the hydrophobic fatty acyl chain of a medium-chain-length ((3R)-OH-C10) substrate as bent into the binding pocket, whereas the short-chain substrates are dislocated by two mechanisms: (i) a short-chain-length 3-hydroxyacyl group ((3R)-OH-C4) does not reach the hydrophobic contacts needed for anchoring the substrate into the active site; and (ii) Leu44 in the loop above the NAD(+) cofactor attracts short-chain-length substrates away from the active site. Dehydrogenase B, which can use a (3R)-OH-C4 substrate, has a more shallow binding pocket and the substrate is correctly placed for catalysis. Based on the current structure, and together with the structure of the 2-enoyl-CoA hydratase 2 unit of yeast MFE-2 it becomes obvious that in yeast and mammalian MFE-2s, despite basically identical functional domains, the assembly of these domains into a mature, dimeric multifunctional enzyme is very different.
Subject(s)
3-Hydroxyacyl CoA Dehydrogenases/chemistry , 3-Hydroxyacyl CoA Dehydrogenases/metabolism , Candida tropicalis/enzymology , Peroxisomes/enzymology , 3-Hydroxyacyl CoA Dehydrogenases/genetics , Amino Acid Sequence , Animals , Candida tropicalis/genetics , Crystallography, X-Ray , Dimerization , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Species Specificity , Static Electricity , Substrate SpecificityABSTRACT
D-bifunctional protein (DBP) deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation. The clinical presentation of DBP deficiency is usually very severe, but a few patients with a relatively mild presentation have been identified. In this article, we report the mutational spectrum of DBP deficiency on the basis of molecular analysis in 110 patients. We identified 61 different mutations by DBP cDNA analysis, 48 of which have not been reported previously. The predicted effects of the different disease-causing amino acid changes on protein structure were determined using the crystal structures of the (3R)-hydroxyacyl-coenzyme A (CoA) dehydrogenase unit of rat DBP and the 2-enoyl-CoA hydratase 2 unit and liganded sterol carrier protein 2-like unit of human DBP. The effects ranged from the replacement of catalytic amino acid residues or residues in direct contact with the substrate or cofactor to disturbances of protein folding or dimerization of the subunits. To study whether there is a genotype-phenotype correlation for DBP deficiency, these structure-based analyses were combined with extensive biochemical analyses of patient material (cultured skin fibroblasts and plasma) and available clinical information on the patients. We found that the effect of the mutations identified in patients with a relatively mild clinical and biochemical presentation was less detrimental to the protein structure than the effect of mutations identified in those with a very severe presentation. These results suggest that the amount of residual DBP activity correlates with the severity of the phenotype. From our data, we conclude that, on the basis of the predicted effect of the mutations on protein structure, a genotype-phenotype correlation exists for DBP deficiency.