Why does mitochondrial complex I have so many subunits?

The prokaryotic and eukaryotic homologues of complex I (proton-pumping NADH:quinone oxidoreductase) perform the same function in energy transduction, but the eukaryotic enzymes are twice as big as their prokaryotic cousins, and comprise three times as many subunits. Fourteen core subunits are conserved in all complexes I, and are sufficient for catalysis - so why are the eukaryotic enzymes embellished by so many supernumerary or accessory subunits? In this issue of the Biochemical Journal, Angerer et al. have provided new evidence to suggest that the supernumerary subunits are important for enzyme stability. This commentary aims to put this suggestion into context.     

Relatedness of archaebacterial RNA polymerase core subunits to their eubacterial and eukaryotic equivalents.

The sequence of the genes encoding the four largest subunits of the RNA polymerase of the archaebacterium Methanobacterium thermoautotrophicum was determined and putative translation signals were identified. The genes are more strongly homologous to eukaryotic than to eubacterial RNA polymerase genes. Analysis of the polypeptide sequences revealed colinearity of two pairs of adjacent archaebacterial genes encoding the B" and B' or A and C genes, respectively, with two eubacterial and two eukaryotic genes each encoding the two largest RNA polymerase subunits. This difference in sequence organization is discussed in terms of gene fusion in the course of evolution. The degree of conservation is much higher between the archaebacterial and the eukaryotic polypeptides than between the archaebacterial and the eubacterial enzyme. Putative functional domains were identified in two of the subunits of the archaebacterial enzyme.     

Cytochrome c oxidase: structure, function, and membrane topology of the polypeptide subunits.

Mitochondrial cytochrome c oxidase and its bacterial homologs catalyze electron transfer and proton translocation reactions across membranes. The eukaryotic enzyme complex consists of a large number of polypeptide subunits. Three of the subunits (I, II, and III) are mitochondrially encoded while the remaining 6 (yeast) to 10 (bovine) are nuclear encoded. Antibody and chemical-labelling experiments suggest that subunits I-III and most (but not all) of the nuclear-encoded subunits span the inner mitochondrial membrane. Subunits I and II are the catalytic core of the enzyme. Subunit I contains haem a, haem a3 and CuB, while subunit II contains CuA and the cytochrome c binding site. Subunit III and most of the nuclear subunits are essential for the assembly of a functional catalytic enzyme. Some nuclear subunits are present as isozymes, although little functional difference has yet been detected between enzyme complexes composed of different isozymes. Therefore, any additional role attributed to the nuclear-encoded subunits beyond that of enzyme assembly must be tentative. We suggest that enough evidence exists to support the idea that modification of the larger nuclear subunits (IV, V, and possibly VI) can effect enzyme turnover in vitro. Whether this is a physiological control mechanism remains to be seen.     

Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis.

Cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The T. novellus enzyme is composed of two kinds of subunits of 32,000 and 23,000 daltons and its minimum molecular weight is 55,000 on the basis of heme content and amino acid composition. The N. agilis enzyme also has two kinds of subunits of 40,000 and 27,000 daltons and its minimum molecular weight is 66,000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.     

Reconstitution and functional characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania donovani

Leishmania donovani topoisomerase I is an unusual bi-subunit enzyme. The activity of the enzyme has been detected when the genes of the individual subunits were co-expressed in yeast [J. Biol. Chem. 278 (2003) 3521]. Here, we report for the first time, the in vitro reconstitution of the two recombinant proteins, LdTOP1L and LdTOP1S, corresponding to the large and small subunits and localization of the active enzyme in both the nucleus and kinetoplast. The proteins were purified from bacterial extract and the activity was measured by plasmid DNA relaxation assay. LdTOP1L and LdTOP1S form a direct 1:1 heterodimer complex through protein-protein interaction. Under standard relaxation assay condition (50 mM KCl and 10 mM Mg(2+)), reconstituted enzyme (LdTOP1LS) showed reduced processivity as well as 2-fold reduced affinity for DNA compared to eukaryotic monomeric rat liver topoisomerase I (RLTOP1). Cleavage assay at various salt concentrations reveals that Camptothecin (CPT) enhanced the formation of "cleavable complex" at low salt. Interaction between the two subunits leading to the formation of an active complex could be explored as an insight for development of new therapeutic agents with specific selectivity.     

Structure and evolution of prokaryotic and eukaryotic RNA polymerases: a model

A comparative overview of the subunit taxonomy and sequences of eukaryotic and prokaryotic RNA polymerases indicates the presence of a core structure conserved between both sets of enzymes. The differentiation between prokaryotic and eukaryotic polymerases is ascribed to domains and subunits peripheral to the largely conserved central structure. Possible subunit and domain functions are outlined. The core's flexible shape is largely determined by the elongated architecture of the two largest subunits, which can be oriented along the DNA axis with their bulkier amino-terminal head regions looking towards the 3' end of the gene to be transcribed and their more slender carboxyl-terminal domains at the tail end of the enzyme. The two largest prokaryotic subunits appear originally derived from a single gene.     

Characterization of enzymatic properties of human ribonucleotide reductase holoenzyme reconstituted in vitro from hRRM1, hRRM2, and p53R2 subunits

Ribonucleotide reductase (RR) is a highly regulated enzyme in the deoxyribonucleotide synthesis pathway. RR is responsible for the de novo conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, which are essential for DNA synthesis and repair. Besides two subunits, hRRM1 and hRRM2, p53R2 is a newly identified member of RR family that is induced by ultraviolet light in a p53-dependent manner. To understand the molecular interaction of RR subunits, we employed a eukaryotic expression system to express and purify all three subunits. After in vitro reconstitution, the results of [(3)H]CDP reduction assay showed that both eukaryotic recombinant hRRM2 and p53R2 proteins could interact with hRRM1 to form functional RR holoenzyme. The reconstituted RR activity was time-dependent and the reaction rate reached the plateau phase after 40min incubation. No matter the concentration, RR holoenzyme reconstituted from p53R2 and hRRM1 could only achieve about 40-75% kinetic activity of that from hRRM2 and hRRM1. The synthetic C-terminal heptapeptide competition assays confirmed that hRRM2 and p53R2 share the same binding site on hRRM1, but the binding site on hRRM1 demonstrated higher affinity for hRRM2 than for p53R2. In allosteric regulation assay, the effect of activation or inhibition of hRRM1 with ATP or dATP suggested that these effectors could regulate RR activity independent of different RR small subunits. Taken together, the eukaryotic expression system RR holoenzyme will provide a very useful tool to understand the molecular mechanisms of RR activity and the interactions of its subunits.     

High Conformational Stability of Secreted Eukaryotic Catalase-peroxidases: ANSWERS FROM FIRST CRYSTAL STRUCTURE AND UNFOLDING STUDIES

Catalase-peroxidases (KatGs) are bifunctional heme enzymes widely spread in archaea, bacteria, and lower eukaryotes. Here we present the first crystal structure (1.55 Å resolution) of an eukaryotic KatG, the extracellular or secreted enzyme from the phytopathogenic fungus Magnaporthe grisea. The heme cavity of the homodimeric enzyme is similar to prokaryotic KatGs including the unique distal ⁺Met-Tyr-Trp adduct (where the Trp is further modified by peroxidation) and its associated mobile arginine. The structure also revealed several conspicuous peculiarities that are fully conserved in all secreted eukaryotic KatGs. Peculiarities include the wrapping at the dimer interface of the N-terminal elongations from the two subunits and cysteine residues that cross-link the two subunits. Differential scanning calorimetry and temperature- and urea-mediated unfolding followed by UV-visible, circular dichroism, and fluorescence spectroscopy combined with site-directed mutagenesis demonstrated that secreted eukaryotic KatGs have a significantly higher conformational stability as well as a different unfolding pattern when compared with intracellular eukaryotic and prokaryotic catalase-peroxidases. We discuss these properties with respect to the structure as well as the postulated roles of this metalloenzyme in host-pathogen interactions.     

Lack of assembly of mitochondrial DNA-encoded subunits of respiratory NADH dehydrogenase and loss of enzyme activity in a human cell mutant lacking the mitochondrial ND4 gene product.

In most eukaryotic cells, the respiratory chain NADH dehydrogenase (Complex I) is a multimeric enzyme under dual (nuclear and mitochondrial) genetic control. Several genes encoding subunits of this enzyme have been identified in the mitochondrial genome from various organisms, but the functions of these subunits are in most part unknown. We describe here a human cell line in which the enzyme lacks the mtDNA-encoded subunit ND4 due to a frameshift mutation in the gene. In this cell line, the other mtDNA-encoded subunits fail to assemble, while at least some of the nuclear-encoded subunits involved in the redox reactions appear to be assembled normally. In fact, while there is a complete loss of NADH:Q1 oxidoreductase activity, the NADH:Fe(CN)6 oxidoreductase activity is normal. These observations provide the first clear evidence that the ND4 gene product is essential for Complex I activity and give some insights into the function and the structural relationship of this polypeptide to the rest of the enzyme. They are also significant for understanding the pathogenetic mechanism of the ND4 gene mutation associated with Leber's hereditary optic neuropathy.     

Expression and characterization of recombinant human acyloxyacyl hydrolase, a leukocyte enzyme that deacylates bacterial lipopolysaccharides

The molecular cloning and eukaryotic cell expression of the complementary DNA for human neutrophil acyloxyacyl hydrolase (AOAH) are described. AOAH is a leukocyte enzyme that selectively removes the secondary (acyloxyacyl-linked) fatty acyl chains from the lipid A region of bacterial lipopolysaccharides (endotoxins), thereby detoxifying the molecules. The two disulfide-linked subunits of the enzyme are encoded by a single mRNA. The amino acid sequence of the protein contains a lipase consensus sequence in the large subunit and a region in the small subunit that is similar to the saposins, cofactors for sphingolipid hydrolases. The recombinant enzyme, like native AOAH, hydrolyzes secondary acyl chains from more than one position on the lipopolysaccharide backbone. Acyloxyacyl hydrolase is a novel two-component lipase that, by deacylating lipopolysaccharides, may modulate host inflammatory responses to Gram-negative bacterial invasion.     

Evolutionary aspects of cytochromec oxidase

The presence of additional subunits in cytochrome oxidase distinguish the multicellular eukaryotic enzyme from that of a simple unicellular bacterial enzyme. The number of these additional subunits increases with increasing evolutionary stage of the organism. Subunits I–III of the eukaryotic enzyme are related to the three bacterial subunits, and they are encoded on mito-chondrial DNA. The additional subunits are nuclear encoded. Experimental evidences are presented here to indicate that the lower enzymatic activity of the mammalian enzyme is due to the presence of nuclear-coded subunits. Dissociation of some of the nuclear-coded subunits (e.g., VIa) by laurylmaltoside and anions increased the activity of the rat liver enzyme to a value similar to that of the bacterial enzyme. Further, it is shown that the intraliposomal nucleotides influence the kinetics of ferrocytochromec oxidation by the reconstituted enzyme from bovine heart but not fromP. denitrificans. The regulatory function attributed to the nuclear-coded subunits of mammalian cytochromec oxidase is also demonstrated by the tissue-specific response of the reconstituted enzyme from bovine heart but not from bovine liver to intraliposomal ADP. These enzymes from bovine heart and liver differ in the amino acid sequences of subunits VIa, VIIa, and VIII. The results presented here are taken to indicate a regulation of cytochromec oxidase activity by nuclear-coded subunits which act like receptors for allosteric effectors and influence the catalytic activity of the core enzyme via conformational changes.     

Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit

Protein kinase CK2 (formerly called: casein kinase 2) is a heterotetrameric enzyme composed of two separate catalytic chains (CK2alpha) and a stable dimer of two non-catalytic subunits (CK2beta). CK2alpha is a highly conserved member of the superfamily of eukaryotic protein kinases. The crystal structure of a C-terminal deletion mutant of human CK2alpha was solved and refined to 2.5A resolution. In the crystal the CK2alpha mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix alphaC and the activation segment, two main regions of conformational plasticity and regulatory importance in eukaryotic protein kinases, in active conformations stabilized by extensive contacts to the N-terminal segment. This arrangement is in accordance with the constitutive activity of the enzyme. By structural superimposition of human CK2alpha in isolated form and embedded in the human CK2 holoenzyme the loop connecting the strands beta4 and beta5 and the ATP-binding loop were identified as elements of structural variability. This structural comparison suggests that the ATP-binding loop may be the key region by which the non-catalytic CK2beta dimer modulates the activity of CK2alpha. The beta4/beta5 loop was found in a closed conformation in contrast to the open conformation observed for the CK2alpha subunits of the CK2 holoenzyme. CK2alpha monomers with this closed beta4/beta5 loop conformation are unable to bind CK2beta dimers in the common way for sterical reasons, suggesting a mechanism to protect CK2alpha from integration into CK2 holoenzyme complexes. This observation is consistent with the growing evidence that CK2alpha monomers and CK2beta dimers can exist in vivo independently from the CK2 holoenzyme and may possess physiological roles of their own.     

The structure of the carboxyltransferase component of acetyl-coA carboxylase reveals a zinc-binding motif unique to the bacterial enzyme

Acetyl-coA carboxylase (ACC) is a central metabolic enzyme that catalyzes the committed step in fatty acid biosynthesis: biotin-dependent conversion of acetyl-coA to malonyl-coA. The bacterial carboxyltransferase (CT) subunit of ACC is a target for the design of novel therapeutics that combat severe, hospital-acquired infections resistant to the established classes of frontline antimicrobials. Here, we present the structures of the bacterial CT subunits from two prevalent nosocomial pathogens, Staphylococcus aureus and Escherichia coli, at a resolution of 2.0 and 3.0 A, respectively. Both structures reveal a small, independent zinc-binding domain that lacks a complement in the primary sequence or structure of the eukaryotic homologue.     

Nuclear and cytoplasmic genome relationships in the genus Avena: Analysis by isoelectric focusing of ribulose biphosphate carboxylase subunits

Comparisons of the isoelectric points of small and large subunits of ribulose biphosphate carboxylase extracted from a number of diploid, tetraploid, and hexaploid Avena species have been used to obtain information on the nuclear and cytoplasmic genome relationships within the genus. All species tested had small subunits with similar isoelectric points, so their analysis provided no information of taxonomic value. Three types of large subunits could be distinguished by this method, and the distribution of each among the available species provides strong evidence against the involvement of a C genome diploid (such as A. ventricosa) as the maternal parent in the formation of either tetraploid or hexaploid species. One type of large subunit was confined to the perennial tetraploid, A. macrostachya, and its position in the genus and possible origin are discussed. The value of this approach in studying genome relationships within the genus Avena and related genera is assessed.     

Manipulations in the peripheral stalk of the Saccharomyces cerevisiae F1F0-ATP synthase

The Saccharomyces cerevisiae F(1)F(0)-ATP synthase peripheral stalk is composed of the OSCP, h, d, and b subunits. The b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of F(1). In contrast, the Escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional F(1)F(0)-ATP synthase. The differences in subunit structure between the eukaryotic and prokaryotic peripheral stalks raised a question about whether the two stalks have similar physical and functional properties. In the present work, the length of the S. cerevisiae b subunit has been manipulated to determine whether the F(1)F(0)-ATP synthase exhibited the same tolerances as in the bacterial enzyme. Plasmid shuffling was used for ectopic expression of altered b subunits in a strain carrying a chromosomal disruption of the ATP4 gene. Wild type growth phenotypes were observed for insertions of up to 11 and a deletion of four amino acids on a nonfermentable carbon source. In mitochondria-enriched fractions, abundant ATP hydrolysis activity was seen for the insertion mutants. ATPase activity was largely oligomycin-insensitive in these mitochondrial fractions. In addition, very poor complementation was seen in a mutant with an insertion of 14 amino acids. Lengthier deletions yielded a defective enzyme. The results suggest that although the eukaryotic peripheral stalk is near its minimum length, the b subunit can be extended a considerable distance.     

Tracing the evolution of a large protein complex in the eukaryotes, NADH:ubiquinone oxidoreductase (Complex I)

The increasing availability of sequenced genomes enables the reconstruction of the evolutionary history of large protein complexes. Here, we trace the evolution of NADH:ubiquinone oxidoreductase (Complex I), which has increased in size, by so-called supernumary subunits, from 14 subunits in the bacteria to 30 in the plants and algae, 37 in the fungi and 46 in the mammals. Using a combination of pair-wise and profile-based sequence comparisons at the levels of proteins and the DNA of the sequenced eukaryotic genomes, combined with phylogenetic analyses to establish orthology relationships, we were able to (1) trace the origin of six of the supernumerary subunits to the alpha-proteobacterial ancestor of the mitochondria, (2) detect previously unidentified homology relations between subunits from fungi and mammals, (3) detect previously unidentified subunits in the genomes of several species and (4) document several cases of gene duplications among supernumerary subunits in the eukaryotes. One of these, a duplication of N7BM (B17.2), is particularly interesting as it has been lost from genomes that have also lost Complex I proteins, making it a candidate for a Complex I interacting protein. A parsimonious reconstruction of eukaryotic Complex I evolution shows an initial increase in size that predates the separation of plants, fungi and metazoa, followed by a gradual adding and incidental losses of subunits in the various evolutionary lineages. This evolutionary scenario is in contrast to that for Complex I in the prokaryotes, for which the combination of several separate, and previously independently functioning modules into a single complex has been proposed.     

Solution structure of the N-terminal domain of the archaeal D-family DNA polymerase small subunit reveals evolutionary relationship to eukaryotic B-family polymerases

Archaea-specific D-family DNA polymerase forms a heterotetramer consisting of two large polymerase subunits and two small exonuclease subunits. We analyzed the structure of the N-terminal 200 amino-acid regulatory region of the small subunit by NMR and revealed that the N-terminal ∼70 amino-acid region is folded. The structure consists of a four-α-helix bundle including a short parallel β-sheet, which is similar to the N-terminal regions of the B subunits of human DNA polymerases α and ε, establishing evolutionary relationships among these archaeal and eukaryotic polymerases. We observed monomer-dimer equilibrium of this domain, which may be related to holoenzyme architecture and/or functional regulation.