Dehydrogenase Fragment Research Articles

Characterization of Two Quinone Radicals in the NADH:Ubiquinone Oxidoreductase from Escherichia coli by a Combined Fluorescence Spectroscopic and Electrochemical Approach

The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. It was proposed that the electron transfer involves quinoid groups localized at the end of the electron transfer chain. To identify these groups, fluorescence excitation and emission spectra of Escherichia coli complex I and its fragments, namely, the NADH dehydrogenase fragment containing the flavin mononucleotide and six iron-sulfur (Fe-S) clusters, and the quinone reductase fragment containing three Fe-S clusters were measured. Signals sensitive to reduction by either NADH or dithionite were detected within the complex and the quinone reductase fragment and attributed to the redox transition of protonated ubiquinone radicals. A fluorescence spectroscopic electrochemical redox titration revealed midpoint potentials of -37 and- 235 mV (vs the standard hydrogen electrode) for the redox transitions of the quinone radicals in complex I at pH 6 with an absorption around 325 nm and a fluorescence emission at 460/475 nm. The role of these cofactor(s) for electron transfer is discussed.

On the molecular basis of D-bifunctional protein deficiency type III.

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 (Km, Vmax and kcat) 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.

Disruption of individual nuo-genes leads to the formation of partially assembled NADH:ubiquinone oxidoreductase (complex I) in Escherichia coli

The proton-pumping NADH:ubiquinone oxidoreductase, respiratory complex I, couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. In Escherichia coli the complex is made up of 13 different subunits encoded by the so-called nuo-genes. Mutants, in which each of the nuo-genes was individually disrupted by the insertion of a resistance cartridge were unable to assemble a functional complex I. Each disruption resulted in the loss of complex I-mediated activity and the failure to extract a structurally intact complex. Thus, all nuo-genes are required either for the assembly or the stability of a functional E. coli complex I. The three subunits comprising the soluble NADH dehydrogenase fragment of the complex were detected in the cytoplasm of several nuo-mutants as one distinct band after BN-PAGE. It is discussed that the fully assembled NADH dehydrogenase fragment represents an assembly intermediate of the E. coli complex I. A partially assembled complex I bound to the membrane was detected in the nuoK and nuoL mutants, respectively. Overproduction of the ΔNuoL variant resulted in the accumulation of two populations of a partially assembled complex in the cytoplasmic membranes. Both populations are devoid of NuoL. One population is enzymatically active, while the other is not. The inactive population is missing cluster N2 and is tightly associated with the inducible lysine decarboxylase. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Nucleotide-induced conformational changes in the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The energy-converting NADH:ubiquinone oxidoreductase, also known as respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex consisting of a peripheral and a membrane arm. The peripheral arm contains all known cofactors and the NADH-binding site, whereas the membrane arm has to be involved in proton translocation. Owing to this, a conformation-linked mechanism for redox-driven proton translocation is discussed. By means of electron microscopy, we show that both arms of the Escherichia coli complex I are widened after the addition of NADH but not of NADPH. NADH-induced conformational changes were also detected in solution: ATR-FTIR (attenuated total reflection Fourier-transform infrared) of the soluble NADH dehydrogenase fragment of the complex indicates protein re-arrangements induced by the addition of NADH. EPR spectroscopy of surface mutants of the complex containing a covalently bound spin label at distinct positions demonstrates NADH-dependent conformational changes in both arms of the complex.

S4.21 Monitoring the conformational change of the NADH dehydrogenase fragment from the E. coli complex I by attenuated total reflexion spectroscopy

S4.21 Monitoring the conformational change of the NADH dehydrogenase fragment from the E. coli complex I by attenuated total reflexion spectroscopy

Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR

BackgroundCandida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence) gene family can be performed using quantitative PCR (qPCR). In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans) biofilms, using the geNorm Visual Basic Application (VBA) for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts.ResultsThe eight genes tested in this study are ranked according to their expression stability (from most stable to least stable) as follows: ACT1 (β-actin)/PMA1 (adenosine triphosphatase), RIP (ubiquinol cytochrome-c reductase complex component), RPP2B (cytosolic ribosomal acidic protein P2B), LSC2 (succinyl-CoA synthetase β-subunit fragment), IMH3 (inosine-5'-monophosphate dehydrogenase fragment), CPA1 (carbamoyl-phosphate synthethase small subunit) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase).Our data indicate that five genes are necessary for accurate and reliable normalization of gene expression data in C. albicans biofilms. Using different normalization strategies, we found a significant upregulation of the ALS1 gene and downregulation of the ALS3 gene in C. albicans biofilms grown on silicone disks in a continous flow system, the CDC reactor (Centre for Disease Control), for 24 hours.ConclusionIn conclusion, we recommend the use of the geometric mean of the relative expression values from the five housekeeping genes (ACT1, PMA1, RIP, RPP2B and LSC2) for normalization, when analysing differences in gene expression levels between C. albicans biofilm cells and planktonic cells. Validation of the normalization strategies described above showed that the ALS1 gene is overexpressed and the ALS3 gene is underexpressed in C. albicans biofilms grown on silicone in the CDC reactor for 24 hours.

Gene Disruption of Spred-2 Causes Dwarfism

The impact of the fibroblast growth factor receptor 3 (FGFR3)-mediated signaling pathway on bone growth has been demonstrated by various genetic approaches. Overexpression of fibroblast growth factors (FGFs), several gain-of-function mutations in the FGFR3, and constitutive activation of mitogen-activated protein kinase (MAPK) kinase (MEK1) in chondrocytes have been shown to cause dwarfism in mice by activation of the MAPK signaling pathway. To investigate the previously reported inhibitory role of Spred in the FGFR3/MAPK pathway, we generated mice with a trapped Spred-2 gene. Here we show that lack of functional Spred-2 protein in mice caused a dwarf phenotype, similar to achondroplasia, the most common form of human dwarfism. Spred-2(-/-) mice showed reduced growth and body weight, they had a shorter tibia length, and showed narrower growth plates as compared with wild-type mice. We detected promoter activity and protein expression of Spred-2 in chondrocytes, suggesting an important function of Spred-2 in chondrocytes and bone development. Stimulation of chondrocytes with different FGF concentrations showed earlier and augmented ERK phosphorylation in Spred-2(-/-) chondrocytes in comparison to Spred-2(+/+) chondrocytes. Our observations suggest a model in which loss of Spred-2 inhibits bone growth by inhibiting chondrocyte differentiation through up-regulation of the MAPK signaling pathway.

EPR Signals Assigned to Fe/S Cluster N1c of the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I) Derive from Cluster N1a

The proton-pumping NADH:ubiquinone oxidoreductase, which is also called respiratory complex I, transfers electrons from NADH to ubiquinone via one flavin mononucleotide (FMN) and up to nine iron-sulfur clusters. A structural minimal form of complex I consisting of 14 different subunits called NuoA to NuoN (or Nqo1 to Nqo14) is found in bacteria. The isolated Escherichia coli complex I can be split into a NADH dehydrogenase fragment, a connecting fragment, and a membrane fragment. The soluble NADH dehydrogenase fragment represents the electron input part of the complex and consists of the subunits NuoE, F, and G. The FMN and four iron-sulfur clusters have been detected in this fragment by means of EPR spectroscopy. One of the EPR signals, called N1c, has spectral properties, which are not found in preparations of the complex from other organisms. Therefore, it is attributed to an additional binding motif on NuoG, which is present only in a few bacteria including E. coli. Here, we show by means of EPR spectroscopic analysis of the NADH dehydrogenase fragment containing site-directed mutations on NuoG that the EPR signals in question derived from cluster N1a on NuoE. The mutations in NuoG disturbed the assembly of the overproduced NADH dehydrogenase fragment indicating that a yet undetected cluster might be bound to the additional motif. Thus, there is no third binuclear iron-sulfur "N1c" in the E. coli complex I but an additional tetranuclear cluster that may be coined N7.

Mechanism of the Reductive Half-reaction in Cellobiose Dehydrogenase

The extracellular flavocytochrome cellobiose dehydrogenase (CDH; EC ) participates in lignocellulose degradation by white-rot fungi with a proposed role in the early events of wood degradation. The complete hemoflavoenzyme consists of a catalytically active dehydrogenase fragment (DH(cdh)) connected to a b-type cytochrome domain via a linker peptide. In the reductive half-reaction, DH(cdh) catalyzes the oxidation of cellobiose to yield cellobiono-1,5-lactone. The active site of DH(cdh) is structurally similar to that of glucose oxidase and cholesterol oxidase, with a conserved histidine residue positioned at the re face of the flavin ring close to the N5 atom. The mechanisms of oxidation in glucose oxidase and cholesterol oxidase are still poorly understood, partly because of lack of experimental structure data or difficulties in interpreting existing data for enzyme-ligand complexes. Here we report the crystal structure of the Phanerochaete chrysosporium DH(cdh) with a bound inhibitor, cellobiono-1,5-lactam, at 1.8-A resolution. The distance between the lactam C1 and the flavin N5 is only 2.9 A, implying that in an approximately planar transition state, the maximum distance for the axial 1-hydrogen to travel for covalent addition to N5 is 0.8-0.9 A. The lactam O1 interacts intimately with the side chains of His-689 and Asn-732. Our data lend substantial structural support to a reaction mechanism where His-689 acts as a general base by abstracting the O1 hydroxyl proton in concert with transfer of the C1 hydrogen as hydride to the re face of the flavin N5.

An assessment of the pathways of dark reduction and oxidation of the plastoquinone pool in thylakoid membranes of higher plants and green algae

Several possible `chlororespiratory? pathways have been proposed for both reduction and oxidation of the plastoquinone pool in the dark in cells of both higher plants and green algae. However, bioinformatic analysis of the recently published genome of Arabidopsis thaliana sets limits to the types of respiratory-related components that could be present in higher plant thylakoids: for example, homologues of cytochrome c oxidase, succinate dehydrogenase and alternative NDH-2 enzymes in thylakoids can be ruled out and there are also no thylakoid-targetted homologues of the NADH-reactive fragment of mitochondrial complex I (the 75kDa, 51 kDa and 30 kDa subunits) that might provide an NADH dehydrogenase fragment for the other complex I homologues that are encoded in the chloroplast genome. In parallel to these considerations, direct assay of pathways of dark reduction and oxidation of plastoquinone has been made in intact leaves and in cells of Chlamydomonas reinhardtii by monitoring P700+ reduction, the slow phase of the carotenoid bandshift and turnover kinetics of the cytochrome bf complex. Combination of these measurements with effects of a wide range of specific inhibitors of various quinone-reactive sites has allowed further definition of the types of pathways and their extents.

FT-IR spectroscopic characterization of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli: oxidation of FeS cluster N2 is coupled with the protonation of an aspartate or glutamate side chain.

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. It couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. One FMN and up to nine iron-sulfur (FeS) clusters participate in the redox reaction. So far, complex I has been described mainly by means of EPR- and UV-vis spectroscopy. Here, we report for the first time an infrared spectroscopic characterization of complex I. Electrochemically induced FT-IR difference spectra of complex I from Escherichia coli and of the NADH dehydrogenase fragment of this complex were obtained for critical potential steps. The spectral contributions of the FMN in both preparations were derived from a comparison using model compounds and turned out to be unexpectedly small. Furthermore, the FT-IR difference spectra reveal that the redox transitions of the FMN and of the FeS clusters induce strong reorganizations of the polypeptide backbone. Additional signals in the spectra of complex I reflect contributions induced by the redox transition of the high-potential FeS cluster N2 which is not present in the NADH dehydrogenase fragment. Part of these signals are attributed to the reorganization of protonated/deprotonated Asp or Glu side chains. On the basis of these data we discuss the role of N2 for proton translocation of complex I.

The F420H2 dehydrogenase from Methanosarcina mazei is a Redox-driven proton pump closely related to NADH dehydrogenases.

The F(420)H(2) dehydrogenase is part of the energy conserving electron transport system of the methanogenic archaeon Methanosarcina mazei Gö1. Here it is shown that cofactor F(420)H(2)-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H(+) translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + P(i). The gene cluster encoding the F(420)H(2) dehydrogenase of M. mazei Gö1 comprises 12 genes that are referred to as fpoA, B, C, D, H, I, J, K, L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F(420)H(2) dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei Gö1. Instead, the gene product FpoF may be responsible for F(420)H(2) oxidation and may function as the electron input part. Thus, the F(420)H(2) dehydrogenase from M. mazei Gö1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the methanogenic archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F(420) and a modified output module adopted to the reduction of methanophenazine.

One-step purification of the NADH dehydrogenase fragment of the Escherichia coli complex I by means of Strep-tag affinity chromatography

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Complex I of Escherichia coli can be split into three fragments. One of these fragments, the soluble NADH dehydrogenase fragment, represents the electron input part of complex I. It comprises the subunits NuoE, F and G and harbors one flavin mononucleotide and up to six iron-sulfur clusters. Here, we report the one-step purification of this fragment by means of affinity chromatography on StrepTactin. This was achieved by fusing the Strep-tag II peptide to the C-terminus of NuoF or NuoG. Fusion of this peptide to the N-terminus of either NuoE or NuoF disturbed the assembly of the NADH dehydrogenase fragment.

Characterization of the overproduced NADH dehydrogenase fragment of the NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli.

The proton-pumping NADH:ubiquinone oxidoreductase of Escherichia coli is composed of 14 different subunits and contains one FMN and up to nine iron-sulfur clusters as prosthetic groups. By use of salt treatment, the complex can be split into an NADH dehydrogenase fragment, a connecting fragment and a membrane fragment. The water-soluble NADH dehydrogenase fragment has a molecular mass of approximately 170,000 Da and consists of the subunits NuoE, F, and G. The fragment harbors the FMN and probably six iron-sulfur clusters, four of them being observable by EPR spectroscopy. Here, we report that the fully assembled fragment can be overproduced in E. coli when the genes nuoE, F, and G were simultaneously overexpressed with the genes nuoB, C, and D. Furthermore, riboflavin, sodium sulfide, and ferric ammonium citrate have to be added to the culture medium. The fragment was purified from the cytoplasm by means of ammonium sulfate fractionation and chromatographic steps. The preparation contains one noncovalently bound FMN per molecule. Two binuclear (N1b and N1c) and two tetranuclear (N3 and N4) iron-sulfur clusters were detected by EPR in the NADH reduced preparation with spectral characteristics identical with those of the corresponding clusters in complex I. The preparation fulfills all prerequisites for crystallization of the fragment.

Structure of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.

When D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein was purified from human liver, two preparations were obtained. One contained a 77-kDa polypeptides as the main and minor smaller polypeptides including a 46-kDa polypeptide, and this preparation showed both the dehydratase and dehydrogenase activities. The other preparation was a homodimer of the 46-kDa polypeptide and showed only the dehydratase activity. Further analysis indicated that the native enzyme is a homodimer of 77-kDa polypeptide, which was proteolytically modified during purification. The cDNA for the human 77-kDa polypeptide was cloned. The amino acid sequences of the peptides derived from the components of the enzyme preparations were located in the deduced amino acid sequence of the cDNA. The preparation containing the 77-kDa polypeptide was treated with a protease, and two monofunctional fragments were separated. The dehydrogenase and dehydratase fragments were located on the amino- and carboxyl-terminal sides, respectively, of the deduced amino acid sequence of the cDNA. The protein expressed by the cDNA with the entire coding region exhibited both the dehydratase and dehydrogenase activities, and that expressed by a truncated version covering the carboxyl-terminal side exhibited only the dehydratase activity. The cloned cDNA was identical to the human 17 beta-hydroxysteroid dehydrogenase IV cDNA.

Isolation and Characterization of the Proton-translocating NADH:ubiquinone Oxidoreductase from Escherichia coli

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) was isolated from Escherichia coli by chromatographic steps performed in the presence of an alkylglucoside detergent at pH 6.0. The complex is obtained in a monodisperse state with a molecular mass of approximately 550,000 Da and is composed of 14 subunits. The subunits were assigned to the 14 genes of the nuo operon, partly based on their N-terminal sequences and partly on their apparent molecular masses. The preparation contains one noncovalently bound FMN/molecule. At least two binuclear (N1b and N1c) and three tetranuclear (N2, N3 and N4) iron-sulfur clusters were detected by EPR in the preparation when reduced with NADH. Their EPR characteristics remained mostly unaltered during the isolation process. After reconstitution in phospholipid membranes, the preparation catalyses piericidin-A-sensitive electron transfer from NADH to ubiquinone-2 with Km values similar to those of complex I in cytoplasmic membranes but with only 10% of the Vmax value. The isolated complex I was cleaved into three fragments when the pH was raised from 6.0 to 7.5 and the detergent exchanged to Triton X-100. One of these fragments is a water-soluble NADH dehydrogenase fragment which is composed of three subunits bearing at least four iron-sulfur clusters (N1b, N1c, N3 and N4) that can be reduced with NADH, one of them bearing FMN. The second, amphipathic, fragment, which is presumed to connect the NADH dehydrogenase fragment with the membrane, contains four subunits and at least one EPR-detectable iron-sulfur cluster whose spectral properties are reminiscent of the eucaryotic cluster N2. The third membrane fragment is composed of seven homologues of the mitochondrially encoded subunits of the eucaryotic complex I. This subunit arrangement coincidences to some extent with the order of the genes on the nuo operon. A topological model of the E. coli complex I is proposed.

Isolation and Characterization of the Proton-translocating NADH:ubiquinone Oxidoreductase from Escherichia coli

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) was isolated from Escherichia coli by chromatographic steps performed in the presence of an alkylglucoside detergent at pH 6.0. The complex is obtained in a monodisperse state with a molecular mass of approximately 550,000 Da and is composed of 14 subunits. The subunits were assigned to the 14 genes of the nuo operon, partly based on their N-terminal sequences and partly on their apparent molecular masses. The preparation contains one noncovalently bound FMN/molecule. At least two binuclear (N1b and N1c) and three tetranuclear (N2, N3 and N4) iron-sulfur clusters were detected by EPR in the preparation when reduced with NADH. Their EPR characteristics remained mostly unaltered during the isolation process. After reconstitution in phospholipid membranes, the preparation catalyses piericidin-A-sensitive electron transfer from NADH to ubiquinone-2 with Km values similar to those of complex I in cytoplasmic membranes but with only 10% of the Vmax value. The isolated complex I was cleaved into three fragments when the pH was raised from 6.0 to 7.5 and the detergent exchanged to Triton X-100. One of these fragments is a water-soluble NADH dehydrogenase fragment which is composed of three subunits bearing at least four iron-sulfur clusters (N1b, N1c, N3 and N4) that can be reduced with NADH, one of them bearing FMN. The second, amphipathic, fragment, which is presumed to connect the NADH dehydrogenase fragment with the membrane, contains four subunits and at least one EPR-detectable iron-sulfur cluster whose spectral properties are reminiscent of the eucaryotic cluster N2. The third membrane fragment is composed of seven homologues of the mitochondrially encoded subunits of the eucaryotic complex I. This subunit arrangement coincidences to some extent with the order of the genes on the nuo operon. A topological model of the E. coli complex I is proposed.

Characterization of proteolysis fragments of aspartokinase I: homoserine dehydrogenase I. Fluorescence and circular dichroism studies.

Proteolysis of native aspartokinase-homoserine dehydrogenase by chymotrypsin, subtilisin, clostripain, and V8 protease yields active dehydrogenase fragments. Fluorescence and near-UV circular dichroism measurements demonstrate that the bulk of the spectroscopic signal observed in the native protein originates in the residual fragments. Kinetic studies and far-UV CD spectra further distribute the fragments into two groups. Even though the remaining dehydrogenase activity is no longer inhibited by L-threonine, ultrafiltration binding studies and far-UV CD spectra clearly demonstrate that one of the two sets of inhibitor-binding sites is still intact. Computer analysis of the far-UV CD data of the native protein and the isolated fragments in the presence and absence of L-threonine has been used to resolve contributions from helix, beta, turn, and aperiodic components. This analysis indicates that the binding of the inhibitor induces decreases in helix content and generation of aperiodic structure within the molecule. The changes observed are similar in the native molecule and the fragments.

Two regions of the bifunctional protein aspartokinase I- homoserine dehydrogenase I are connected by a short hinge.

Four proteases differing in their specificity, i.e. subtilisin, trypsin, alpha-chymotrypsin and V8 staphylococcal protease, cleave the bifunctional protein Escherichia coli aspartokinase I-homoserine dehydrogenase I (composed of 820 residues) producing an active homoserine dehydrogenase fragment. This cleavage occurs within a short segment of the polypeptide chain extending from residue 293 to residue 300.

Immunochemical comparison of a bifunctional enzyme, aspartokinase II-homoserine dehydrogenase II, and its two proteolytic fragments

We report here a comparison between immunochemical properties of the bifunctional enzyme aspartokinase II-homoserine dehydrogenase II of E.coli K12 and of its two isolated proteolytic fragments. Both fragments, one inactive and one endowed with homoserine dehydrogenase activity, react with antibodies raised against the native enzyme. Some of the antibodies elicited against the dehydrogenase fragment can recognize regions of this fragment which are not exposed in the entire enzyme. The immunochemical results are used to discuss a simple model in which this bifunctional enzyme is folded up in two domains. The organization of aspartokinase II-homoserine dehydrogenase II is compared to that of another bifunctional enzyme aspartokinase I-homoserine dehydrogenase I with which it shares some sequence homology.