Molybdenum Cofactor

Overview

The Molybdenum Cofactor (MoCo) is a catalytically active protein which incorporates the element Molybdenum.

The biosynthesis of MoCo involves the complex interaction of six proteins and is a process of four steps, which also require iron, ATP, and copper. After its synthesis, Moco is distributed, involving MoCo-binding proteins.

Role in Health

MoCo deficiency results in a severe inborn error of metabolism causing often early childhood death. Disease-causing symptoms mainly go back to the lack of sulfite oxidase (SO) activity, an enzyme in cysteine catabolism. Besides their name-giving functions, Mo-enzymes have been recognized to catalyze novel reactions, including the reduction of nitrite to nitric oxide. (R2)

“Under conditions of Moco or sulphite oxidase deficiency, sulphite accumulates in plasma and serum, crosses the blood–brain barrier and rapidly triggers neuronal death.

Impaired ATP synthesis has been suggested as one possible mechanism of sulphite toxicity.

Sulphite accumulation also triggers the reduction of cystine, the main carrier for cysteine in serum and plasma, which consequently causes the formation of S-sulphocysteine, a potential agonist of glutamate receptors.

The latter may explain the observed seizures, convulsions, contractions and twitching associated with MoCD, causing damage of cortical neurons as documented by abnormal magnetic resonance imaging of the brain and loss of white matter.” (R4)

S-sulfocysteine is agonist of NMDA receptors

Here, we studied S-sulfocysteine (SSC), a structural analog of glutamate that accumulates in the plasma and urine of patients with MoCD, and demonstrated that it acts as an N-methyl d-aspartate receptor (NMDA-R) agonist, leading to calcium influx and downstream cell signaling events and neurotoxicity.

SSC treatment activated the protease calpain, and calpain-dependent degradation of the inhibitory synaptic protein gephyrin subsequently exacerbated SSC-mediated excitotoxicity and promoted loss of GABAergic synapses.

Pharmacological blockade of NMDA-R, calcium influx, or calpain activity abolished SSC and glutamate neurotoxicity in primary murine neurons. Finally, the NMDA-R antagonist memantine was protective against the manifestation of symptoms in a tungstate-induced MoCD mouse model.

These findings demonstrate that SSC drives excitotoxic neurodegeneration in MoCD and introduce NMDA-R antagonists as potential therapeutics for this fatal disease. (R12)

Patients with MoCD have high levels of sulfite in their urine, which is accompanied by elevated urinary levels of taurine and thiosulfate, while their plasma cystine level is depleted.

In addition, S-sulfocysteine (SSC), a secondary sulfur-containing metabolite, accumulates in the urine and plasma of MoCD patients and is used for MoCD diagnostics. (R12)

Metabolic anomalies

Low serum and urine Uric Acid (R10)
Presenсe of sulfite in Urine (R10)

Sulfite Oxidase abundance depends on MoCo

These results confirm the past results showing that SO is degraded in the absence of Moco which is evident in MOCS3 KO cells and a slight reduction of SO abundance in TUM1 KO cells.

The low amount of SO activity present in the TUM1 KO cells might be due to sulfur availability for MOCS3 transferred from other sulfurtransferases. (R9)

Dependencies

MoCo synthesis

MoCo synthesis depends on:

Copper (see Biosynthesis section)
Iron (as 4Fe-4S clusters) - MOCS1 (source UniProtKB).
SAMe - MOCS1 is a radical SAMe enzyme (source UniProtKB).
Zinc - MOCS3 (source UniProtKB).
Magnesium - GPHN (source UniProtKB).
L-cysteine - MOCOS (source UniProtKB) and NFS1 (UniprotKB).
pyridoxal 5'-phosphate - MOCOS (source UniProtKB) and NFS1 (UniprotKB).

Enzymes using MoCo

Sulphite Oxidase (gene SUOX)
Xanthine Oxidase (gene XDH)
Aldehyde Oxidase (gene AOX1)
Mitochondrial amidoxime-reducing component 1 (gene MTARC1)
Mitochondrial amidoxime-reducing component 2 (gene MTARC2)

Cellular Uptake

Molybdate enters cells via specific high affinity transport system. Also through the sulfate and phosphate uptake system (at least in bacteria). (R3)

Biosynthesis

Moco is chemically unstable especially under aerobic conditions, and cannot be taken up as a nutrient. Thus, Moco must be synthesized de novo in cells, unlike many other cofactors which can be taken up as nutrients (R13)

As with biopterin, the synthesis begins with GTP. However, the first two enzymes of the pathway convert GTP to an intermediate containing a four-membered ring, cyclic pyranopterin monophosphate, or cPMP.

The first of these enzymes utilizes S-adenosylmethionine in a radical reaction.

A copper atom and sulfurs are added to cPMP at the position that will eventually contain molybdenum, in a reaction that yields MPT, or metal-binding pterin.

In humans an enzyme named gephyrin accomplishes the last steps.

Gephyrin adenylates the molecule, and inserts molybdenum. AMP is then lost, giving the Moco molecule. (R7)

Dependency on Iron-Sulfur clusters

Molybdenum metabolism is tightly connected to Fe-S cluster synthesis in that some of the molybdenum enzymes and Moco biosynthesis itself depend on Fe-S enzymes and on a mitochondrial transporter that is known to be crucial for the maturation of cytosolic Fe-S proteins.

Moreover, Moco biosynthesis recruits mechanisms previously known from Fe-S cluster synthesis, which involves the mobilization of sulfide for the formation of a Mo-S center for specific molybdenum enzymes, which will be touched upon below. (R1)

and another paper suggests this openly:

since CNX2 and MOCS1A require Fe-S clusters for activity, it is reasonable to assume that a Moco-deficient phenotype may also be caused by a deficiency in the biosynthesis of Fe-S clusters.

In support of this, Arabidopsis lines with downregulated levels of NFS1, the mitochondrial Cys desulfurase with an essential role in Fe-S cluster biosynthesis, had strongly decreased AO activities (Frazzon et al., 2007). However, whether this is due to decreased activity of CNX2 or to Fe-S assembly on cytosolic AO proteins was not investigated. (R14)

Step 1. Synthesis of cPMP (precursor Z) from GTP

Iron-sulfur cluster dimer MOCS1A+MOCS1AB produces cyclic PMP, Methionine, PPi and 5'-deoxyadenosine from GTP, H2O and SAMe. This reaction happens in the mitochondria (R1).

cPMP is the most stable intermediate of Moco biosynthesis, with an estimated half-life of several hours at low pH (R1)

PMP = Pyranopterin monophosphate.

Export of cPMP from mitochondria to cytosol

After the synthesis of cPMP in the mitochondria it is transported from to cytosol via a complex that contains ABCB7:

In contrast to the first step, all subsequent steps of Moco biosynthesis were demonstrated to be localized in the cytosol.

Although cPMP is hydrophobic enough to pass through biological membranes simply by diffusion, recent work on plants demonstrated that a specific transporter in the inner membrane of mitochondria is involved in the transport of cPMP into the cytosol. (R1)

As the accumulation of cPMP in mitochondria seems to be a direct consequence of the ATM3 mutation, our data indicate that ATM3 is involved in the export of cPMP from mitochondria into the cytosol. These findings provide evidence for a novel function of mitochondria and the mitochondrial ABC half-transporter ATM3 in Moco biosynthesis. (R14)

ATM3 in plants (Atm1p in yeast) is a homolog of ABCB7 in humans.

Interestingly, together with yeast Atm1p and mammalian ABCB7, this transporter was originally identified to be essential for the maturation of extramitochondrial Fe-S proteins by transporting an as yet unknown compound generated during mitochondrial Fe-S cluster synthesis into the cytosol, where it serves as a substrate for the cytosolic Fe-S assembly machinery. (R1)

Since both 2Fe-2S clusters and MPT are transported by this transporter, we can assume there can be a competition of substrates, that affect MoCo synthesis efficiency during iron deficiency treatment.

What is also interesting, 2Fe-2S clusters are transported bound by four molecules of GSH (R15, Uniprot). Which leads to a loss of GSH inside mitochondria.

Step 2. cPMP transformed to MPT (molybdopterin)

First, MOCS2 dimer is loaded with two sulfur atoms by MOCS3 (sulfur is cleaved from cysteine by NFS1).

Second, MOCS2A transfers sulfur to cPMP (precursor Z).

Third, MOCS2B cleaves the ring (deadenylation), producing MPT.

After the reaction, MOCS2 complex needs to be resulfurated again by MOCS3 (see the first sentence in this section).

At this step, Copper is added to the protein. TODO: Find how exactly.

Step 3. MPT is adenylated, forming MPT-AMP

Proteins: Gephyrin (GPHN) as a trimeric complex Reaction: MPT + AMP = MPT-AMP within the Gephyrin protein.

In step 3, MPT is bound by the two-domain proteins, CNX1 in plants and GEPHYRIN in humans, and is activated by adenylation, whereby MPT-AMP is formed (Kuper et al., 2004). This reaction is restricted to the G-domains of these proteins and is assumed to prepare MPT for insertion of molybdenum. (R14)

Step 4. Molybdenum atom is inserted into MPT

Proteins: Gephyrin (GPHN) as a trimeric complex Reaction: MoO₄ + MPT-AMP = MoCo + copper + AMP Cofactors: Mg²⁺

During the fourth and final step, MPT-AMP is transferred to the E-domains of CNX1 and GEPHYRIN, respectively, where the adenylate is cleaved and molybdenum is inserted to finally yield mature Moco. (R14)

Step 5 (optional). MoCo is sulfurated by MOCOS

MOCOS (P5P enzyme) sulfurates MoCo using cysteine, so that sulfurated MoCo can be used in xanthine oxidase and aldehyde oxidaze (SUOX doesn’t require sulfuration). (Reactome)

MPT synthase Associating Complex (MTPAC)

Cytoplasmic MPT Synthase (MOCS3) forms a complex with several other proteins: DBN1, CLNS1A, SNRPD2, SNRPB/B’, and HSD17B10:

MPT synthase is found in a 274 kDa cytoplasmic complex called the MPT synthase associating complex (MPTAC), which contains MOCS3, DBN1, CLNS1A, SNRPD2, SNRPB/B’, and HSD17B10. (R16).

DBN1

CLNS1A

SNRPD2

SNRPB

HSD17B10

MPTAC associates with PRMT5 and SNRP

The association of MPTAC with Protein arginine (R) Methyltransferase 5 (PRMT5) complex and small nuclear ribonucleoprotein (SNRP) splicing factors enables SNRPs to sense metabolic states through their methylation.

This promotes the splicing fidelity of amyloid precursor protein (APP) pre-mRNA and proper APP fragmentation, abnormalities of which have been observed in the platelets of AD patients.

The functions of MPTAC are crucial to maintain expression of drebrin 1, which is required for synaptic plasticity, through prevention from oxidative damage. Thus, adjustment of sulfur amino acid catabolism by MPTAC prevents events that occur early in the onset of AD. (R17)

MOCS2 inhibition leads to ROS accumulation

The depletion of glucose and pyruvate led to decreases in SAMe and increases in the population of apoptotic cells in MOCS2 knockdown cells but not in control cells.

Hence, inhibited sulfur amino acid catabolism by MOCS2 knockdown engages ROS accumulation. (R17)

MPTAC regulates Fatty acid oxidation

The levels of malonylcarnitine were significantly increased in MOCS2 knockdown cells. Malonylcarnitine belongs to acyl carnitines and accumulates with disruption of fatty acid oxidation, which is caused by the failed entry of long-chain acylcarnitine esters into the mitochondria and by deficiency of malonyl-CoA decarboxylase (MLYCD), which recycles malonyl-CoA to acetyl-CoA.

RNA levels of MLYCD decreased in MOCS2 knockdown cells (Figure 2A). Malonylcarnitine also accumulates with a deficiency of acyl-CoA dehydrogenase, catalyzing fatty acid β-oxidation.

Importantly, MPTAC contains HSD17B10, which catalyzes β-oxidation. (!)

RNA levels of carnitine palmitoyltransferase 1/2 (CPT1/2), acyl-CoA dehydrogenase (ACADM), enol-CoA hydratase (EHHADH), HSD17B10, and 3-ketoacyl-CoA dehydrogenase (HADHB) were reduced in MOCS2 knockdown cells.

Reduction of HSD17B10 RNA levels was more sensitive than that of its protein levels in MOCS2 knockdown cells.

Hence, MPTAC is required for fatty acid oxidation. Since cells utilize fatty acid oxidation to support energy needs in the absence of glucose, the roles of MPTAC in fatty acid oxidation may engage to prevent ROS accumulations upon glucose and pyruvate depletion (R17)

Genes

MOCS1 - Molybdenum cofactor biosynthesis protein 1. Defects in this gene lead to “Molybdenum cofactor deficiency, type A”. (UniProtKB). Two proteins are made from this gene: MOCS1A and MOCS1AB via alternative splicing (R11).

MOCS2 - Molybdopterin synthase sulfur carrier subunit. Acts as a sulfur carrier required for molybdopterin biosynthesis. Component of the molybdopterin synthase complex that catalyzes the conversion of precursor Z into molybdopterin by mediating the incorporation of 2 sulfur atoms into precursor Z to generate a dithiolene group. (UniProtKB)

MOCS3 - Adenylyltransferase and sulfurtransferase MOCS3. The protein encoded by this gene adenylates and activates molybdopterin synthase, an enzyme required for biosynthesis of MoCo. This gene contains no introns.

MOCOS - Molybdenum cofactor sulfurase. Sulfurates the molybdenum cofactor which is required for activation of the xanthine dehydrogenase (XDH) and aldehyde oxidase (AO) enzymes. Defects in this gene cause the metabolic disorder “classical xanthinuria type II” which is characterized by the loss of XDH/XO and AO enzyme activity, decreased levels of uric acid in the urine, increased levels of xanthine and hypoxanthine in the serum and urine, formation of xanthine stones in the urinary tract, and myositis due to tissue deposition of xanthine.

GPHN - Gephyrin (other name “Molybdopterin molybdenumtransferase”). Catalyzes two steps in the biosynthesis of the molybdenum cofactor. In the first step, molybdopterin is adenylated. Subsequently, molybdate is inserted into adenylated molybdopterin and AMP is released.

Gephyrin is a central player at inhibitory postsynapses, directly binds and organizes GABA-A and glycine receptors (GABAARs and GlyRs), and is thereby indispensable for normal inhibitory neurotransmission. (R5)

MFSD5 - Molybdate-anion transporter. Mediates high-affinity intracellular uptake of the rare oligo-element molybdenum. (R6])

NFS1 - Cysteine desulfurase. Appears to provide sulfur from Cysteine to MOCS3 (R8).

MPST - mercaptopyruvate sulfurtransferase. Interacts with NFS1 and MOCS3 (R?)

ABCB7 - mitochondrial exporter of Iron-Sulfur clusters and cPMP. (R1, R14)

Interactions

Copper

Excessive consumption of Molybdenum may lead to copper deficiency:

“excessive fertilization, resulting in molybdenum overload of the soil that caused pathological symptoms of molybdenosis in animals; this, in particular in ruminants, triggered secondary copper deficiency” [R4]

Gephyrin is inhibited by Copper and Tungsten (source UniProtKB).