AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5.

The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long-lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.

Biallelic gephyrin variants lead to impaired GABAergic inhibition in a patient with developmental and epileptic encephalopathy.

Synaptic inhibition is essential for shaping the dynamics of neuronal networks, and aberrant inhibition is linked to epilepsy. Gephyrin (Geph) is the principal scaffolding protein at inhibitory synapses and is essential for postsynaptic clustering of glycine (GlyRs) and GABA type A receptors. Consequently, gephyrin is crucial for maintaining the relationship between excitation and inhibition in normal brain function and mutations in the gephyrin gene (GPHN) are associated with neurodevelopmental disorders and epilepsy. We identified bi-allelic variants in the GPHN gene, namely the missense mutation c.1264G > A and splice acceptor variant c.1315-2A > G, in a patient with developmental and epileptic encephalopathy. We demonstrate that the splice acceptor variant leads to nonsense-mediated mRNA decay. Furthermore, the missense variant (D422N) alters gephyrin structure, as examined by analytical size exclusion chromatography and circular dichroism-spectroscopy, thus leading to reduced receptor clustering and sensitivity towards calpain-mediated cleavage. In addition, both alterations contribute to an observed reduction of inhibitory signal transmission in neurons, which likely contributes to the pathological encephalopathy.

Consensus guidelines for the diagnosis and management of isolated sulfite oxidase deficiency and molybdenum cofactor deficiencies.

Sulfite intoxication is the hallmark of four ultrarare disorders that are caused by impaired sulfite oxidase activity due to genetic defects in the synthesis of the molybdenum cofactor or of the apoenzyme sulfite oxidase. Delays on the diagnosis of these disorders are common and have been caused by their unspecific presentation of acute neonatal encephalopathy with high early mortality, followed by the evolution of dystonic cerebral palsy and also by the lack of easily available and reliable diagnostic tests. There is significant variation in survival and in the quality of symptomatic management of affected children. One of the four disorders, molybdenum cofactor deficiency type A (MoCD-A) has recently become amenable to causal treatment with synthetic cPMP (fosdenopterin). The evidence base for the rational use of cPMP is very limited. This prompted the formulation of these clinical guidelines to facilitate diagnosis and support the management of patients. The guidelines were developed by experts in diagnosis and treatment of sulfite intoxication disorders. It reflects expert consensus opinion and evidence from a systematic literature search.

Molecular mechanism of intramolecular electron transfer in dimeric sulfite oxidase.

Sulfite oxidase (SOX) is a homodimeric molybdoheme enzyme that oxidizes sulfite to sulfate at the molybdenum center. Following substrate oxidation, molybdenum is reduced and subsequently regenerated by two sequential electron transfers (ETs) via heme to cytochrome c. SOX harbors both metals in spatially separated domains within each subunit, suggesting that domain movement is necessary to allow intramolecular ET. To address whether one subunit in a SOX dimer is sufficient for catalysis, we produced heterodimeric SOX variants with abolished sulfite oxidation by replacing the molybdenum-coordinating and essential cysteine in the active site. To further elucidate whether electrons can bifurcate between subunits, we truncated one or both subunits by deleting the heme domain. We generated three SOX heterodimers: (i) SOX/Mo with two active molybdenum centers but one deleted heme domain, (ii) SOX/Mo_C264S with one unmodified and one inactive subunit, and (iii) SOX_C264S/Mo harboring a functional molybdenum center on one subunit and a heme domain on the other subunit. Steady-state kinetics showed 50% SOX activity for the SOX/Mo and SOX/Mo_C264S heterodimers, whereas SOX_C264S/Mo activity was reduced by two orders of magnitude. Rapid reaction kinetics monitoring revealed comparable ET rates in SOX/Mo, SOX/Mo_C264S, and SOX/SOX, whereas in SOX_C264S/Mo, ET was strongly compromised. We also combined a functional SOX Mo domain with an inactive full-length SOX R217W variant and demonstrated interdimer ET that resembled SOX_C264S/Mo activity. Collectively, our results indicate that one functional subunit in SOX is sufficient for catalysis and that electrons derived from either Mo(IV) or Mo(V) follow this path.

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.

Alternative splicing of the bicistronic gene molybdenum cofactor synthesis 1 (MOCS1) uncovers a novel mitochondrial protein maturation mechanism.

Molybdenum cofactor (Moco) biosynthesis is a highly conserved multistep pathway. The first step, the conversion of GTP to cyclic pyranopterin monophosphate (cPMP), requires the bicistronic gene molybdenum cofactor synthesis 1 (MOCS1). Alternative splicing of MOCS1 within exons 1 and 9 produces four different N-terminal and three different C-terminal products (type I-III). Type I splicing results in bicistronic transcripts with two open reading frames, of which only the first, MOCS1A, is translated, whereas type II/III splicing produces MOCS1AB proteins. Here, we first report the cellular localization of alternatively spliced human MOCS1 proteins. Using fluorescence microscopy, fluorescence spectroscopy, and cell fractionation experiments, we found that depending on the alternative splicing of exon 1, type I splice variants (MOCS1A) either localize to the mitochondrial matrix (exon 1a) or remain cytosolic (exon 1b). MOCS1A proteins required exon 1a for mitochondrial translocation, but fluorescence microscopy of MOCS1AB variants (types II and III) revealed that they were targeted to mitochondria independently of exon 1 splicing. In the latter case, cell fractionation experiments displayed that mitochondrial matrix import was facilitated via an internal motif overriding the N-terminal targeting signal. Within mitochondria, MOCS1AB underwent proteolytic cleavage resulting in mitochondrial matrix localization of the MOCS1B domain. In conclusion, MOCS1 produces two functional proteins, MOCS1A and MOCS1B, which follow different translocation routes before mitochondrial matrix import for cPMP biosynthesis involving both proteins. MOCS1 protein maturation provides a novel alternative splicing mechanism that ensures the coordinated mitochondrial targeting of two functionally related proteins encoded by a single gene.

Impaired mitochondrial maturation of sulfite oxidase in a patient with severe sulfite oxidase deficiency.

Sulfite oxidase (SO) is encoded by the nuclear SUOX gene and catalyzes the final step in cysteine catabolism thereby oxidizing sulfite to sulfate. Oxidation of sulfite is dependent on two cofactors within SO, a heme and the molybdenum cofactor (Moco), the latter forming the catalytic site of sulfite oxidation. SO localizes to the intermembrane space of mitochondria where both-pre-SO processing and cofactor insertion-are essential steps during SO maturation. Isolated SO deficiency (iSOD) is a rare inborn error of metabolism caused by mutations in the SUOX gene that lead to non-functional SO. ISOD is characterized by rapidly progressive neurodegeneration and death in early infancy. We diagnosed an iSOD patient with homozygous mutation of SUOX at c.1084G>A replacing Gly362 to serine. To understand the mechanism of disease, we expressed patient-derived G362S SO in Escherichia coli and surprisingly found full catalytic activity, while in patient fibroblasts no SO activity was detected, suggesting differences between bacterial and human expression. Moco reconstitution of apo-G362S SO was found to be approximately 90-fold reduced in comparison to apo-WT SO in vitro. In line, levels of SO-bound Moco in cells overexpressing G362S SO were significantly reduced compared to cells expressing WT SO providing evidence for compromised maturation of G362S SO in cellulo. Addition of molybdate to culture medium partially rescued impaired Moco binding of G362S SO and restored SO activity in patient fibroblasts. Thus, this study demonstrates the importance of the orchestrated maturation of SO and provides a first case of Moco-responsive iSOD.

Machine learning-based identification and characterization of 15 novel pathogenic SUOX missense mutations.

Isolated sulfite oxidase deficiency (ISOD) is a rare hereditary metabolic disease caused by absence of functional sulfite oxidase (SO) due to mutations of the SUOX gene. SO oxidizes toxic sulfite and sulfite accumulation is associated with neurological disorders, progressive brain atrophy and early death. Similarities of these neurological symptoms to abundant diseases like neonatal encephalopathy underlines the raising need to increase the awareness for ISOD. Here we report an interdisciplinary approach utilizing exome/genome data derived from gnomAD database as well as published variants to predict the pathogenic outcome of 303 naturally occurring SO missense variants and combining these with activity determination. We identified 15 novel ISOD-causing SO variants and generated a databank of pathogenic SO missense variants to support future diagnosis of ISOD patients. We found six inactive variants (W101G, H118Y, E197K, R217W, S427W, D512Y, Q518R) and seven (D110H, P119S, G121E, G130R, Y140C, R269H, Q396P, R459Q) with severe reduction in activity. Based on the Hardy-Weinberg-equilibrium and the combination of our results with published SO missense and protein truncating variants, we calculated the first comprehensive incidence rate for ISOD of 1 in 1,377,341 births and provide a pathogenicity score to 303 naturally occurring SO missense variants.

Oxygen and nitrite reduction by heme-deficient sulphite oxidase in a patient with mild sulphite oxidase deficiency.

Isolated sulphite oxidase deficiency (iSOD) is an autosomal recessive inborn error in metabolism characterised by accumulation of sulphite, which leads to death in early infancy. Sulphite oxidase (SO) is encoded by the SUOX gene and forms a heme- and molybdenum-cofactor-dependent enzyme localised in the intermembrane space of mitochondria. Within SO, both cofactors are embedded in two separated domains, which are linked via a flexible 11 residue tether. The two-electron oxidation of sulphite to sulphate occurs at the molybdenum active site. From there, electrons are transferred via two intramolecular electron transfer steps (IETs) via the heme cofactor and to the physiologic electron acceptor cytochrome c. Previously, we reported nitrite and oxygen to serve as alternative electron acceptors at the Moco active site, thereby overcoming IET within SO. Here, we present evidence for these reactions to occur in an iSOD patient with an unusual mild disease representation. In the patient, a homozygous c.427C>A mutation within the SUOX gene leads to replacement of the highly conserved His143 to Asn. The affected His143 is one of two heme-iron-coordinating residues within SO. We demonstrate, that the H143N SO variant fails to bind heme in vivo leading to the elimination of SO-dependent cytochrome c reduction in mitochondria. We show, that sulphite oxidation at the Moco domain is unaffected in His143Asn SO variant and demonstrate that nitrite and oxygen are able to serve as electron acceptors for sulphite-derived electrons in cellulo. As result, the patient H143N SO variant retains residual sulphite oxidising activity thus ameliorating iSOD progression.

Mechanism of nitrite-dependent NO synthesis by human sulfite oxidase.

In addition to nitric oxide (NO) synthases, molybdenum-dependent enzymes have been reported to reduce nitrite to produce NO. Here, we report the stoichiometric reduction in nitrite to NO by human sulfite oxidase (SO), a mitochondrial intermembrane space enzyme primarily involved in cysteine catabolism. Kinetic and spectroscopic studies provide evidence for direct nitrite coordination at the molybdenum center followed by an inner shell electron transfer mechanism. In the presence of the physiological electron acceptor cytochrome c, we were able to close the catalytic cycle of sulfite-dependent nitrite reduction thus leading to steady-state NO synthesis, a finding that strongly supports a physiological relevance of SO-dependent NO formation. By engineering SO variants with reduced intramolecular electron transfer rate, we were able to increase NO generation efficacy by one order of magnitude, providing a mechanistic tool to tune NO synthesis by SO.

Reciprocal regulation of sulfite oxidation and nitrite reduction by mitochondrial sulfite oxidase.

The oxygen-independent nitrate-nitrite-nitric oxide (NO) pathway is considered as a substantial source of NO in mammals. Dietary nitrate/nitrite are distributed throughout the body and reduced to NO by the action of various enzymes. The intermembrane spaced (IMS), molybdenum cofactor-dependent sulfite oxidase (SO) was shown to catalyze such a nitrite reduction. In this study we asked whether the primary function of SO - sulfite oxidation - and its novel function - nitrite reduction - impact each other. First, we utilized benzyl viologen as artificial electron donor to investigate steady state NO synthesis by SO and found fast (kcat = 14 s-1) nitrite reduction of SO full-length and its isolated molybdenum domain at pH 6.5. Next, we determined the impact of nitrite on pre-steady state kinetics in SO catalysis and identified nitrite as a pH-dependent inhibitor of SO reductive and oxidative half reaction. Finally, we report on the time-dependent formation of the paramagnetic Mo(V) species following nitrite reduction and demonstrate that sulfite inhibits nitrite reduction. In conclusion, we propose a pH-dependent reciprocal regulation of sulfite oxidation and nitrite reduction by each substrate, thus facilitating quick responses to hypoxia induced changes in the IMS, which may function in protecting the cell from reactive oxygen species production.

Palmitoylation of gephyrin controls receptor clustering and plasticity of GABAergic synapses.

Postsynaptic scaffolding proteins regulate coordinated neurotransmission by anchoring and clustering receptors and adhesion molecules. Gephyrin is the major instructive molecule at inhibitory synapses, where it clusters glycine as well as major subsets of GABA type A receptors (GABAARs). Here, we identified palmitoylation of gephyrin as an important mechanism of strengthening GABAergic synaptic transmission, which is regulated by GABAAR activity. We mapped palmitoylation to Cys212 and Cys284, which are critical for both association of gephyrin with the postsynaptic membrane and gephyrin clustering. We identified DHHC-12 as the principal palmitoyl acyltransferase that palmitoylates gephyrin. Furthermore, gephyrin pamitoylation potentiated GABAergic synaptic transmission, as evidenced by an increased amplitude of miniature inhibitory postsynaptic currents. Consistently, inhibiting gephyrin palmitoylation either pharmacologically or by expression of palmitoylation-deficient gephyrin reduced the gephyrin cluster size. In aggregate, our study reveals that palmitoylation of gephyrin by DHHC-12 contributes to dynamic and functional modulation of GABAergic synapses.

Mouse model for molybdenum cofactor deficiency type B recapitulates the phenotype observed in molybdenum cofactor deficient patients.

Molybdenum cofactor (MoCo) deficiency is a rare, autosomal-recessive disorder, mainly caused by mutations in MOCS1 (MoCo deficiency type A) or MOCS2 (MoCo deficiency type B) genes; the absence of active MoCo results in a deficiency in all MoCo-dependent enzymes. Patients with MoCo deficiency present with neonatal seizures, feeding difficulties, severe developmental delay, brain atrophy and early childhood death. Although substitution therapy with cyclic pyranopterin monophosphate (cPMP) has been successfully used in both Mocs1 knockout mice and in patients with MoCo deficiency type A, there is currently no Mocs2 knockout mouse and no curative therapy for patients with MoCo deficiency type B. Therefore, we generated and characterized a Mocs2-null mouse model of MoCo deficiency type B. Expression analyses of Mocs2 revealed a ubiquitous expression pattern; however, at the cellular level, specific cells show prominent Mocs2 expression, e.g., neuronal cells in cortex, hippocampus and brainstem. Phenotypic analyses demonstrated that Mocs2 knockout mice failed to thrive and died within 11 days after birth. None of the tested MoCo-dependent enzymes were active in Mocs2-deficient mice, leading to elevated concentrations of purines, such as hypoxanthine and xanthine, and non-detectable levels of uric acid in the serum and urine. Moreover, elevated concentrations of S-sulfocysteine were measured in the serum and urine. Increased levels of xanthine resulted in bladder and kidney stone formation, whereas increased concentrations of toxic sulfite triggered neuronal apoptosis. In conclusion, Mocs2-deficient mice recapitulate the severe phenotype observed in humans and can now serve as a model for preclinical therapeutic approaches for MoCo deficiency type B.

Oxygen reactivity of mammalian sulfite oxidase provides a concept for the treatment of sulfite oxidase deficiency.

Mammalian sulfite oxidase (SO) is a dimeric enzyme consisting of a molybdenum cofactor- (Moco) and haem-containing domain and catalyses the oxidation of toxic sulfite to sulfate. Following sulfite oxidation, electrons are passed from Moco via the haem cofactor to cytochrome c, the terminal electron acceptor. In contrast, plant SO (PSO) lacks the haem domain and electrons shuttle from Moco to molecular oxygen. Given the high similarity between plant and mammalian SO Moco domains, factors that determine the reactivity of PSO towards oxygen, remained unknown. In the present study, we generated mammalian haem-deficient and truncated SO variants and demonstrated their oxygen reactivity by hydrogen peroxide formation and oxygen-consumption studies. We found that intramolecular electron transfer between Moco and haem showed an inverse correlation to SO oxygen reactivity. Haem-deficient SO variants exhibited oxygen-dependent sulfite oxidation similar to PSO, which was confirmed further using haem-deficient human SO in a cell-based assay. This finding suggests the possibility to use oxygen-reactive SO variants in sulfite detoxification, as the loss of SO activity is causing severe neurodegeneration. Therefore we evaluated the potential use of PEG attachment (PEGylation) as a modification method for future enzyme substitution therapies using oxygen-reactive SO variants, which might use blood-dissolved oxygen as the electron acceptor. PEGylation has been shown to increase the half-life of other therapeutic proteins. PEGylation resulted in the modification of up to eight surface-exposed lysine residues of SO, an increased conformational stability and similar kinetic properties compared with wild-type SO.

Neuronal nitric oxide synthase-dependent S-nitrosylation of gephyrin regulates gephyrin clustering at GABAergic synapses.

Gephyrin, the principal scaffolding protein at inhibitory synapses, is essential for postsynaptic clustering of glycine and GABA type A receptors (GABA(A)Rs). Gephyrin cluster formation, which determines the strength of GABAergic transmission, is modulated by interaction with signaling proteins and post-translational modifications. Here, we show that gephyrin was found to be associated with neuronal nitric oxide synthase (nNOS), the major source of the ubiquitous and important signaling molecule NO in brain. Furthermore, we identified that gephyrin is S-nitrosylated in vivo. Overexpression of nNOS decreased the size of postsynaptic gephyrin clusters in primary hippocampal neurons. Conversely, inhibition of nNOS resulted in a loss of S-nitrosylation of gephyrin and the formation of larger gephyrin clusters at synaptic sites, ultimately increasing the number of cell surface expressed synaptic GABA(A)Rs. In conclusion, S-nitrosylation of gephyrin is important for homeostatic assembly and plasticity of GABAergic synapses.

Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity.

14-3-3 proteins represent a family of ubiquitous eukaryotic proteins involved in numerous signal transduction processes and metabolic pathways. One important 14-3-3 target in higher plants is nitrate reductase (NR), whose activity is regulated by different physiological conditions. Intra-molecular electron transfer in NR is inhibited following 14-3-3 binding to a conserved phospho-serine motif located in hinge 1, a surface exposed loop between the catalytic molybdenum and central heme domain. Here we describe a novel 14-3-3 binding site within the NR N-terminus, an acidic motif conserved in NRs of higher plants, which significantly contributes to 14-3-3-mediated inhibition of NR. Deletion or mutation of the N-terminal acidic motif resulted in a significant loss of 14-3-3 mediated inhibition of Ser534 phosphorylated NR-Mo-heme (residues 1-625), a previously established model of NR regulation. Co-sedimentation and crosslinking studies with NR peptides comprising each of the two binding motifs demonstrated direct binding of either peptide to 14-3-3. Surface plasmon resonance spectroscopy disclosed high-affinity binding of 14-3-3ω to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3ω variant retained interaction to the acidic motif, but lost binding to the phospho-hinge motif. To our knowledge, NR is the first enzyme that harbors two independent 14-3-3 binding sites with different affinities, which both need to be occupied by 14-3-3ω to confer full inhibition of NR activity under physiological conditions.

Involvement of the Cys-Tyr cofactor on iron binding in the active site of human cysteine dioxygenase.

Sulfur metabolism has gained increasing medical interest over the last years. In particular, cysteine dioxygenase (CDO) has been recognized as a potential marker in oncology due to its altered gene expression in various cancer types. Human CDO is a non-heme iron-dependent enzyme, which catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid, which is further metabolized to taurine or pyruvate and sulfate. Several studies have reported a unique post-translational modification of human CDO consisting of a cross-link between cysteine 93 and tyrosine 157 (Cys-Tyr), which increases catalytic efficiency in a substrate-dependent manner. However, the reaction mechanism by which the Cys-Tyr cofactor increases catalytic efficiency remains unclear. In this study, steady-state kinetics were determined for wild type CDO and two different variants being either impaired or saturated with the Cys-Tyr cofactor. Cofactor formation in CDO resulted in an approximately fivefold increase in k cat and tenfold increase in k cat/K m over the cofactor-free CDO variant. Furthermore, iron titration experiments revealed an 18-fold decrease in K d of iron upon cross-link formation. This finding suggests a structural role of the Cys-Tyr cofactor in coordinating the ferrous iron in the active site of CDO in accordance with the previously postulated reaction mechanism of human CDO. Finally, we identified product-based inhibition and α-ketoglutarate and glutarate as CDO inhibitors using a simplified well plate-based activity assay. This assay can be used for high-throughput identification of additional inhibitors, which may contribute to understand the functional importance of CDO in sulfur amino acid metabolism and related diseases.

Modified amelogenin is a new and versatile nanomaterial for biomedical applications.

Amelogenin self-assembly is crucial for tooth biomineralization and crystallite enamel orientation. Amelogenin forms stable nanoparticles under physiological conditions. Here, we tested whether the surface properties and binding characteristics of these particles could be modified to enhance amelogenin function as a biomaterial. We evaluated different amelogenin fusion proteins for their ability to form hybrid nanoparticles. As a proof-of-concept, the integrin-binding tripeptide Arg-Gly-Asp (RGD) sequence from fibronectin was integrated into mouse amelogenin (rM179) at three different positions. Dynamic light scattering (DLS) measurements revealed that these amelogenin fusion proteins still form nanospheres. Additional DLS and isothermal titration calorimetry measurements showed that the mixtures of RGD-modified amelogenin and wild-type amelogenin form stable particles. We determined that insertion of the RGD-loop at the amelogenin C-terminus converts the nanoparticle into a cell-binding substrate. Calvarial osteoblasts efficiently attached and spread on modified amelogenin, whereas almost no binding was observed on wild-type amelogenin. These results establish amelogenin as a new versatile biomaterial that can be easily modified to add additional functions.

Cofactor-dependent maturation of mammalian sulfite oxidase links two mitochondrial import pathways.

Sulfite oxidase (SO) catalyses the metabolic detoxification of sulfite to sulfate within the intermembrane space of mitochondria. The enzyme follows a complex maturation pathway, including mitochondrial transport and processing, integration of two prosthetic groups, molybdenum cofactor (Moco) and heme, as well as homodimerisation. We have identified the sequential and cofactor-dependent maturation steps of SO. The N-terminal bipartite targeting signal of SO was required but not sufficient for mitochondrial localization. In the absence of Moco, most of the SO, although processed by the inner membrane peptidase of mitochondria, was found in the cytosol. Moco binding was required to induce mitochondrial trapping and retention, thus ensuring unidirectional translocation of SO. In the absence of the N-terminal targeting sequence, SO assembled in the cytosol, suggesting an important function for the leader sequence in preventing premature cofactor binding. In vivo, heme binding and dimerisation did not occur in the absence of Moco and only occurred after Moco integration. In conclusion, the identified molecular hierarchy of SO maturation represents a novel link between the canonical presequence pathway and folding-trap mechanisms of mitochondrial import.

Metal insertion into the molybdenum cofactor: product-substrate channelling demonstrates the functional origin of domain fusion in gephyrin.

The complexity of eukaryotic multicellular organisms relies on evolutionary developments that include compartmentalization, alternative splicing, protein domain fusion and post-translational modification. Mammalian gephyrin uniquely exemplifies these processes by combining two enzymatic functions within the biosynthesis of the Moco (molybdenum cofactor) in a multidomain protein. It also undergoes extensive alternative splicing, especially in neurons, where it also functions as a scaffold protein at inhibitory synapses. Two out of three gephyrin domains are homologous to bacterial Moco-synthetic proteins (G and E domain) while being fused by a third gephyrin-specific central C domain. In the present paper, we have established the in vitro Moco synthesis using purified components and demonstrated an over 300-fold increase in Moco synthesis for gephyrin compared with the isolated G domain, which synthesizes adenylylated molybdopterin, and E domain, which catalyses the metal insertion at physiological molybdate concentrations in an ATP-dependent manner. We show that the C domain impacts the catalytic efficacy of gephyrin, suggesting an important structural role in product-substrate channelling as depicted by a structural model that is in line with a face-to-face orientation of both active sites. Our functional studies demonstrate the evolutionary advantage of domain fusion in metabolic proteins, which can lead to the development of novel functions in higher eukaryotes.