Convergent evolution links molybdenum insertase domains with organism-specific sequences.

In all domains of life, the biosynthesis of the pterin-based Molybdenum cofactor (Moco) is crucial. Molybdenum (Mo) becomes biologically active by integrating into a unique pyranopterin scaffold, forming Moco. The final two steps of Moco biosynthesis are catalyzed by the two-domain enzyme Mo insertase, linked by gene fusion in higher organisms. Despite well-understood Moco biosynthesis, the evolutionary significance of Mo insertase fusion remains unclear. Here, we present findings from Neurospora crassa that shed light on the critical role of Mo insertase fusion in eukaryotes. Substituting the linkage region with sequences from other species resulted in Moco deficiency, and separate expression of domains, as seen in lower organisms, failed to rescue deficient strains. Stepwise truncation and structural modeling revealed a crucial 20-amino acid sequence within the linkage region essential for fungal growth. Our findings highlight the evolutionary importance of gene fusion and specific sequence composition in eukaryotic Mo insertases.

Shared functions of Fe-S cluster assembly and Moco biosynthesis.

Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In the recent years it has become evident that the availability of Fe-S clusters play an important role for the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is an enzyme involved in the transfer of sulfur to various acceptor proteins with a main role in the assembly of Fe-S clusters. In this review, we dissect the dependence of the production of active molybdoenzymes in detail, starting from the regulation of gene expression and further explaining sulfur delivery and Fe-S cluster insertion into target enzymes. Further, Fe-S cluster assembly is also linked to iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, we explain that the expression of the genes is dependent on an active FNR protein. FNR is a very important transcription factor that represents the master-switch for the expression of target genes in response to anaerobiosis. Moco biosynthesis is further directly dependent on the presence of ArcA and also on an active Fur protein.

Generation of induced pluripotent stem cells named SMBCi019-A from a methylmalonic acidemia patient carrying the MMACHC mutations.

Methylmalonic acidemia(MMA) is an autosomal recessive hereditary disease caused by methylmalonyl-CoA mutase defect or its coenzyme cobalamin metabolism defect. The mutation of the MMACHC gene leads to metabolic disorder of coenzyme cobalamin, resulting in abnormal accumulation of methylmalonic acid, and finally leads to impairment of multiple organs' functions. Here we generated an induced pluripotent stem cells (iPSCs) line named SMBCi019-A, using urine cells (UCs) derived from a 10-year-old male MMA patient who carried two heterozygous gene mutations in MMACHC c.438G > A (p.w146x) and c.609G > A (p.w203x). The generated iPSCs retained the mutations can function as a cellular model of MMA.

Episodic weakness and axonal sensorimotor neuropathy caused by a mitochondrial MT-ATP6 mutation.

Episodic weakness is typically associated with a group of disorders so called periodic paralyses. Their major causes are mutation of ion channels, and have rarely been linked to mitochondrial disorders. We report a 20-year-old man with episodic weakness and axonal sensorimotor neuropathy since the age of 10 years. Analysis of the next generation sequencing data of the entire mitochondrial genome extracted from the blood revealed a homoplasmic m.9185T > C variant in MT-ATP6. Acetazolamide may be responsive for episodic weakness, and supplements with l-carnitine with coenzyme-Q10 seem to be beneficial as well. To the best of our knowledge, this is the first report in Taiwan which reveals episodic weakness and sensorimotor polyneuropathy as a unique phenotype of MT-ATP6 mutations.

A defect in molybdenum cofactor binding causes an attenuated form of sulfite oxidase deficiency.

Isolated sulfite oxidase deficiency (ISOD) is a rare recessive and infantile lethal metabolic disorder, which is caused by functional loss of sulfite oxidase (SO) due to mutations of the SUOX gene. SO is a mitochondrially localized molybdenum cofactor (Moco)- and heme-dependent enzyme, which catalyzes the vital oxidation of toxic sulfite to sulfate. Accumulation of sulfite and sulfite-related metabolites such as S-sulfocysteine (SSC) are drivers of severe neurodegeneration leading to early childhood death in the majority of ISOD patients. Full functionality of SO is dependent on correct insertion of the heme cofactor and Moco, which is controlled by a highly orchestrated maturation process. This maturation involves the translation in the cytosol, import into the intermembrane space (IMS) of mitochondria, cleavage of the mitochondrial targeting sequence, and insertion of both cofactors. Moco insertion has proven as the crucial step in this maturation process, which enables the correct folding of the homodimer and traps SO in the IMS. Here, we report on a novel ISOD patient presented at 17 months of age carrying the homozygous mutation NM_001032386.2 (SUOX):c.1097G > A, which results in the expression of SO variant R366H. Our studies show that histidine substitution of Arg366, which is involved in coordination of the Moco-phosphate, causes a severe reduction in Moco insertion efficacy in vitro and in vivo. Expression of R366H in HEK SUOX-/- cells mimics the phenotype of patient's fibroblasts, representing a loss of SO expression and specific activity. Our studies disclose a general paradigm for a kinetic defect in Moco insertion into SO caused by residues involved in Moco coordination resulting in the case of R366H in an attenuated form of ISOD.

Change in Cofactor Specificity of Oxidoreductases by Adaptive Evolution of an Escherichia coli NADPH-Auxotrophic Strain.

The nicotinamide cofactor specificity of enzymes plays a key role in regulating metabolic processes and attaining cellular homeostasis. Multiple studies have used enzyme engineering tools or a directed evolution approach to switch the cofactor preference of specific oxidoreductases. However, whole-cell adaptation toward the emergence of novel cofactor regeneration routes has not been previously explored. To address this challenge, we used an Escherichia coli NADPH-auxotrophic strain. We continuously cultivated this strain under selective conditions. After 500 to 1,100 generations of adaptive evolution using different carbon sources, we isolated several strains capable of growing without an external NADPH source. Most isolated strains were found to harbor a mutated NAD+-dependent malic enzyme (MaeA). A single mutation in MaeA was found to switch cofactor specificity while lowering enzyme activity. Most mutated MaeA variants also harbored a second mutation that restored the catalytic efficiency of the enzyme. Remarkably, the best MaeA variants identified this way displayed overall superior kinetics relative to the wild-type variant with NAD+. In other evolved strains, the dihydrolipoamide dehydrogenase (Lpd) was mutated to accept NADP+, thus enabling the pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase complexes to regenerate NADPH. Interestingly, no other central metabolism oxidoreductase seems to evolve toward reducing NADP+, which we attribute to several biochemical constraints, including unfavorable thermodynamics. This study demonstrates the potential and biochemical limits of evolving oxidoreductases within the cellular context toward changing cofactor specificity, further showing that long-term adaptive evolution can optimize enzyme activity beyond what is achievable via rational design or directed evolution using small libraries. IMPORTANCE In the cell, NAD(H) and NADP(H) cofactors have different functions. The former mainly accepts electrons from catabolic reactions and carries them to respiration, while the latter provides reducing power for anabolism. Correspondingly, the ratio of the reduced to the oxidized form differs for NAD+ (low) and NADP+ (high), reflecting their distinct roles. We challenged the flexibility of E. coli's central metabolism in multiple adaptive evolution experiments using an NADPH-auxotrophic strain. We found several mutations in two enzymes, changing the cofactor preference of malic enzyme and dihydrolipoamide dehydrogenase. Upon deletion of their corresponding genes we performed additional evolution experiments which did not lead to the emergence of any additional mutants. We attribute this restricted number of mutational targets to intrinsic thermodynamic barriers; the high ratio of NADPH to NADP+ limits metabolic redox reactions that can regenerate NADPH, mainly by mass action constraints.

Specificity of NifEN and VnfEN for the Assembly of Nitrogenase Active Site Cofactors in Azotobacter vinelandii.

The nitrogen-fixing microbe Azotobacter vinelandii has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their in vivo cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in A. vinelandii that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site. IMPORTANCE Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants.

Seesaw conformations of Npl4 in the human p97 complex and the inhibitory mechanism of a disulfiram derivative.

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.

The requirement for cobalt in vitamin B12: A paradigm for protein metalation.

Vitamin B12, cobalamin, is a cobalt-containing ring-contracted modified tetrapyrrole that represents one of the most complex small molecules made by nature. In prokaryotes it is utilised as a cofactor, coenzyme, light sensor and gene regulator yet has a restricted role in assisting only two enzymes within specific eukaryotes including mammals. This deployment disparity is reflected in another unique attribute of vitamin B12 in that its biosynthesis is limited to only certain prokaryotes, with synthesisers pivotal in establishing mutualistic microbial communities. The core component of cobalamin is the corrin macrocycle that acts as the main ligand for the cobalt. Within this review we investigate why cobalt is paired specifically with the corrin ring, how cobalt is inserted during the biosynthetic process, how cobalt is made available within the cell and explore the cellular control of cobalt and cobalamin levels. The partitioning of cobalt for cobalamin biosynthesis exemplifies how cells assist metalation.

Molybdenum cofactor biology, evolution and deficiency.

The molybdenum cofactor (Moco) represents an ancient metal­sulfur cofactor, which participates as catalyst in carbon, nitrogen and sulfur cycles, both on individual and global scale. Given the diversity of biological processes dependent on Moco and their evolutionary age, Moco is traced back to the last universal common ancestor (LUCA), while Moco biosynthetic genes underwent significant changes through evolution and acquired additional functions. In this review, focused on eukaryotic Moco biology, we elucidate the benefits of gene fusions on Moco biosynthesis and beyond. While originally the gene fusions were driven by biosynthetic advantages such as coordinated expression of functionally related proteins and product/substrate channeling, they also served as origin for the development of novel functions. Today, Moco biosynthetic genes are involved in a multitude of cellular processes and loss of the according gene products result in severe disorders, both related to Moco biosynthesis and secondary enzyme functions.

Production of active recombinant human aldehyde oxidase (AOX) in the baculovirus expression vector system (BEVS) and deployment in a pre-clinical fraction-of-control AOX compound exposure assay.

Human aldehyde oxidase (AOX) has emerged as a key enzyme activity for consideration in modern drug discovery. The enzyme catalyzes the oxidation of a wide variety of compounds, most notably azaheterocyclics that often form the building blocks of small molecule therapeutics. Failure to consider and assess AOX drug exposure early in the drug development cycle can have catastrophic consequences for novel compounds entering the clinic. AOX is a complex molybdopterin-containing iron-sulfur flavoprotein comprised of two identical 150 kDa subunits that has proven difficult to produce in recombinant form, and a commercial source of the purified human enzyme is currently unavailable. Thus, the potential exposure of novel drug development candidates to human AOX metabolism is usually assessed by using extracts of pooled human liver cytosol as a source of the enzyme. This can complicate the assignment of AOX-specific compound exposure due to its low activity and the presence of contaminating enzymes that may have overlapping substrate specificities. Herein is described a two-step process for the isolation of recombinant human AOX dimers to near homogeneity following production in the baculovirus expression vector system (BEVS). The deployment of this BEVS-produced recombinant human AOX as a substitute for human liver extracts in a fraction-of-control AOX compound-exposure screening assay is described. The ability to generate this key enzyme activity readily in a purified recombinant form provides for a more accurate and convenient approach to the assessment of new compound exposure to bona fide AOX drug metabolism.

DNAmoreDB, a database of DNAzymes.

Deoxyribozymes, DNA enzymes or simply DNAzymes are single-stranded oligo-deoxyribonucleotide molecules that, like proteins and ribozymes, possess the ability to perform catalysis. Although DNAzymes have not yet been found in living organisms, they have been isolated in the laboratory through in vitro selection. The selected DNAzyme sequences have the ability to catalyze a broad range of chemical reactions, utilizing DNA, RNA, peptides or small organic compounds as substrates. DNAmoreDB is a comprehensive database resource for DNAzymes that collects and organizes the following types of information: sequences, conditions of the selection procedure, catalyzed reactions, kinetic parameters, substrates, cofactors, structural information whenever available, and literature references. Currently, DNAmoreDB contains information about DNAzymes that catalyze 20 different reactions. We included a submission form for new data, a REST-based API system that allows users to retrieve the database contents in a machine-readable format, and keyword and BLASTN search features. The database is publicly available at https://www.genesilico.pl/DNAmoreDB/.

Identification and characterisation of the Volvox carteri Moco carrier protein.

The molybdenum cofactor (Moco) is a redox active prosthetic group found in the active site of Moco-dependent enzymes (Mo-enzymes). As Moco and its intermediates are highly sensitive towards oxidative damage, these are believed to be permanently protein bound during synthesis and upon maturation. As a major component of the plant Moco transfer and storage system, proteins have been identified that are capable of Moco binding and release but do not possess Moco-dependent enzymatic activities. The first protein found to possess these properties was the Moco carrier protein (MCP) from the green alga Chlamydomonas reinhardtii. Here, we describe the identification and biochemical characterisation of the Volvox carteri (V. carteri) MCP and, for the first time, employ a comparative analysis to elucidate the principles behind MCP Moco binding. Doing so identified a sequence region of low homology amongst the existing MCPs, which we showed to be essential for Moco binding to V. carteri MCP.

Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger.

BACKGROUND: Nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction. RESULTS: We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production. CONCLUSIONS: This data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway (gndA gene) followed by engineering the flux through the reverse TCA cycle (maeA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.

Biological Activity of Pseudovitamin B12 on Cobalamin-Dependent Methylmalonyl-CoA Mutase and Methionine Synthase in Mammalian Cultured COS-7 Cells.

Adenyl cobamide (commonly known as pseudovitamin B12) is synthesized by intestinal bacteria or ingested from edible cyanobacteria. The effect of pseudovitamin B12 on the activities of cobalamin-dependent enzymes in mammalian cells has not been studied well. This study was conducted to investigate the effects of pseudovitamin B12 on the activities of the mammalian vitamin B12-dependent enzymes methionine synthase and methylmalonyl-CoA mutase in cultured mammalian COS-7 cells to determine whether pseudovitamin B12 functions as an inhibitor or a cofactor of these enzymes. Although the hydoroxo form of pseudovitamin B12 functions as a coenzyme for methionine synthase in cultured cells, pseudovitamin B12 does not activate the translation of methionine synthase, unlike the hydroxo form of vitamin B12 does. In the second enzymatic reaction, the adenosyl form of pseudovitamin B12 did not function as a coenzyme or an inhibitor of methylmalonyl-CoA mutase. Experiments on the cellular uptake were conducted with human transcobalamin II and suggested that treatment with a substantial amount of pseudovitamin B12 might inhibit transcobalamin II-mediated absorption of a physiological trace concentration of vitamin B12 present in the medium.

Cofactor Engineering Redirects Secondary Metabolism and Enhances Erythromycin Production in Saccharopolyspora erythraea.

Saccharopolyspora erythraea is used for industrial erythromycin production. To explore the physiological role of intracellular energy state in metabolic regulation by S. erythraea, we initially overexpressed the F1 part of the endogenous F1F0-ATPase in the high yielding erythromycin producing strain E3. The F1-ATPase expression resulted in lower [ATP]/[ADP] ratios, which was accompanied by a strong increase in the production of a reddish pigment and a decreased erythromycin production. Subsequent transcriptional analysis revealed that the lower intracellular [ATP]/[ADP] ratios exerted a pleotropic regulation on the metabolism of S. erythraea. The lower [ATP]/[ADP] ratios induced physiological changes to restore the energy balance, mainly via pathways that tend to produce ATP or regenerate NADH. The F1-ATPase overexpression strain exhibited a state of redox stress, which was correlated to an alteration of electron transport at the branch of the terminal oxidases, and S. erythraea channeled the enhanced glycolytic flux toward a reddish pigment in order to reduce NADH formation. The production of erythromycin was decreased, which is in accordance with the net ATP requirement and the excess NADH formed through this pathway. Partial growth inhibition by apramycin increased the intracellular [ATP]/[ADP] ratios and demonstrated a positive correlation between [ATP]/[ADP] ratios and erythromycin synthesis. Finally, overexpression of the entire F1F0-ATPase complex resulted in 28% enhanced erythromycin production and markedly reduced pigment synthesis in E3. The work illustrates a feasible strategy to optimize the distribution of fluxes in secondary metabolism.

Vitamin and cofactor acquisition in apicomplexans: Synthesis versus salvage.

The Apicomplexa phylum comprises diverse parasitic organisms that have evolved from a free-living ancestor. These obligate intracellular parasites exhibit versatile metabolic capabilities reflecting their capacity to survive and grow in different hosts and varying niches. Determined by nutrient availability, they either use their biosynthesis machineries or largely depend on their host for metabolite acquisition. Because vitamins cannot be synthesized by the mammalian host, the enzymes required for their synthesis in apicomplexan parasites represent a large repertoire of potential therapeutic targets. Here, we review recent advances in metabolic reconstruction and functional studies coupled to metabolomics that unravel the interplay between biosynthesis and salvage of vitamins and cofactors in apicomplexans. A particular emphasis is placed on Toxoplasma gondii, during both its acute and latent stages of infection.

SufT is required for growth of Mycobacterium smegmatis under iron limiting conditions.

Iron-sulphur (FeS) clusters are versatile cofactors required for a range of biological processes within cells. Due to the reactive nature of the constituent molecules, assembly and delivery of these cofactors requires a multi-protein machinery in vivo. In prokaryotes, SufT homologues are proposed to function in the maturation and transfer of FeS clusters to apo-proteins. This study used targeted gene deletion to investigate the role of SufT in the physiology of mycobacteria, using Mycobacterium smegmatis as a model organism. Deletion of the sufT gene in M. smegmatis had no impact on growth under standard culture conditions and did not significantly alter activity of the FeS cluster dependent enzymes succinate dehydrogenase (SDH) and aconitase (ACN). Furthermore, the ΔsufT mutant was no more sensitive than the wild-type strain to the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), or the anti-tuberculosis drugs isoniazid, clofazimine or rifampicin. In contrast, the ΔsufT mutant displayed a growth defect under iron limiting conditions, and an increased requirement for iron during biofilm formation. This data suggests that SufT is an accessory factor in FeS cluster biogenesis in mycobacteria which is required under conditions of iron limitation.

Kinetic and structural evidence that Asp-678 plays multiple roles in catalysis by the quinoprotein glycine oxidase.

PlGoxA from Pseudoalteromonas luteoviolacea is a glycine oxidase that utilizes a protein-derived cysteine tryptophylquinone (CTQ) cofactor. A notable feature of its catalytic mechanism is that it forms a stable product-reduced CTQ adduct that is not hydrolyzed in the absence of O2 Asp-678 resides near the quinone moiety of PlGoxA, and an Asp is structurally conserved in this position in all tryptophylquinone enzymes. In those other enzymes, mutation of that Asp results in no or negligible CTQ formation. In this study, mutation of Asp-678 in PlGoxA did not abolish CTQ formation. This allowed, for the first time, studying the role of this residue in catalysis. D678A and D678N substitutions yielded enzyme variants with CTQ, which did not react with glycine, although glycine was present in the crystal structures in the active site. D678E PlGoxA was active but exhibited a much slower kcat This mutation altered the kinetic mechanism of the reductive half-reaction such that one could observe a previously undetected reactive intermediate, an initial substrate-oxidized CTQ adduct, which converted to the product-reduced CTQ adduct. These results indicate that Asp-678 is involved in the initial deprotonation of the amino group of glycine, enabling nucleophilic attack of CTQ, as well as the deprotonation of the substrate-oxidized CTQ adduct, which is coupled to CTQ reduction. The structures also suggest that Asp-678 is acting as a proton relay that directs these protons to a water channel that connects the active sites on the subunits of this homotetrameric enzyme.

Common Mode of Remodeling AAA ATPases p97/CDC48 by Their Disassembling Cofactors ASPL/PUX1.

The hexameric ring structure of the type II AAA+ ATPases is considered as stable and permanent. Recently, the UBX domain-containing cofactors Arabidopsis thaliana PUX1 and human alveolar soft part sarcoma locus (ASPL) were reported to bind and disassemble the cognate AAA+ ATPases AtCDC48 and human p97. Here, we present two crystal structures related to these complexes: a truncated AtCDC48 (AtCDC48-ND1) and a hybrid complex containing human p97-ND1 and the UBX domain of plant PUX1 (p97-ND1:PUX1-UBX). These structures reveal close similarity between the human and plant AAA+ ATPases, but also highlight differences between disassembling and non-disassembling AAA+ ATPase cofactors. Based on an AtCDC48 disassembly assay with PUX1 and known crystal structures of the p97-bound human cofactor ASPL, we propose a general ATPase disassembly model. Thus, our structural and biophysical investigations provide detailed insight into the mechanism of AAA+ ATPase disassembly by UBX domain cofactors and suggest a general mode of regulating the cellular activity of these molecular machines.