Crystal structure of a molybdopterin synthase-precursor Z complex: insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency

In almost all biological life forms, molybdenum and tungsten are coordinated by molybdopterin (MPT), a tricyclic pyranopterin containing a cis-dithiolene group. Together, the metal and the pterin moiety form the redox reactive molybdenum cofactor (Moco). Mutations in patients with deficiencies in Moco biosynthesis usually occur in the enzymes catalyzing the first and second steps of biosynthesis, leading to the formation of precursor Z and MPT, respectively. The second step is catalyzed by the heterotetrameric MPT synthase protein consisting of two large (MoaE) and two small (MoaD) subunits with the MoaD subunits located at opposite ends of a central MoaE dimer. Previous studies have determined that the conversion of the sulfur- and metal-free precursor Z to MPT by MPT synthase involves the transfer of sulfur atoms from a C-terminal MoaD thiocarboxylate to the C-1' and C-2' positions of precursor Z. Here, we present the crystal structures of non-thiocarboxylated MPT synthase from Staphylococcus aureus in its apo form and in complex with precursor Z. A comparison of the two structures reveals conformational changes in a loop that participates in interactions with precursor Z. In the complex, precursor Z is bound by strictly conserved residues in a pocket at the MoaE dimer interface in close proximity of the C-terminal glycine of MoaD. Biochemical evidence indicates that the first dithiolene sulfur is added at the C-2' position.     

Thiocarboxylation of molybdopterin synthase provides evidence for the mechanism of dithiolene formation in metal-binding pterins.

Molybdopterin (MPT) is a pyranopterin with a unique dithiolene group coordinating molybdenum (Mo) or tungsten (W) in all Mo- and W-enzymes except nitrogenase. In Escherichia coli, MPT is formed by incorporation of two sulfur atoms into precursor Z, which is catalyzed by MPT synthase. The recently solved crystal structure of MPT synthase (Rudolph, M. J., Wuebbens, M. M., Rajagopalan, K. V., and Schindelin, H. (2000) Nat. Struct. Biol. 8, 42-46) shows the heterotetrameric nature of the enzyme that is composed of two small (MoaD) and two large subunits (MoaE). According to sequence and structural similarities among MoaD, ubiquitin, and ThiS, a thiocarboxylation of the C terminus of MoaD is proposed that would serve as the source of sulfur that is transferred to precursor Z. Here, we describe the in vitro generation of carboxylated and thiocarboxylated MoaD. Both forms of MoaD are monomeric and are able to form a heterotetrameric complex after coincubation in equimolar ratios with MoaE. Only the thiocarboxylated MPT synthase complex was found to be able to convert precursor Z in vitro to MPT. Slight but significant differences between the carboxylated and the thiocarboxylated MPT synthase can be seen using size exclusion chromatography. A two-step reaction of MPT synthesis is proposed where the dithiolene is generated by two thiocarboxylates derived from a single tetrameric MPT synthase.     

Cleavage of the moaX-encoded fused molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity

Background

Molybdopterin cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1, moaD2, moaE1, moaE2, the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis.

Results

Using a heterologous Mycobacterium smegmatis host and the activity of the MoCo-dependent nitrate reductase, we confirmed that moaD2 and moaE2 from M. tuberculosis together encode a functional MPT synthase. In contrast, moaD1 displayed no functionality in this system, even in the presence of the MoeBR sulphurtransferase, which contains the rhodansese-like domain, predicted to activate MoaD subunits. We demonstrated that cleavage of MoaX into its constituent MoaD and MoaE subunits was required for MPT synthase activity and confirmed that cleavage occurs between the Gly82 and Ser83 residues in MoaX. Further analysis of the Gly81-Gly82 motif confirmed that both of these residues are necessary for catalysis and that the Gly81 was required for recognition/cleavage of MoaX by an as yet unidentified protease. In addition, the MoaE component of MoaX was able to function in conjunction with M. smegmatis MoaD2 suggesting that cleavage of MoaX renders functionally interchangeable subunits. Expression of MoaX in E. coli revealed that incorrect post-translational processing is responsible for the lack of activity of MoaX in this heterologous host.

Conclusions

There is a degree of functional interchangeability between the MPT synthase subunits of M. tuberculosis. In the case of MoaX, post-translational cleavage at the Gly82 residue is required for function.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0355-2) contains supplementary material, which is available to authorized users.     

Structural studies of molybdopterin synthase provide insights into its catalytic mechanism

Molybdenum cofactor biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea, and eukaryotes, including humans. Genetic deficiencies of enzymes involved in cofactor biosynthesis in humans lead to a severe and usually fatal disease. The molybdenum cofactor contains a tricyclic pyranopterin, termed molybdopterin, that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of molybdopterin is generated by molybdopterin synthase, which consists of a large (MoaE) and small (MoaD) subunit. The crystal structure of molybdopterin synthase revealed a heterotetrameric enzyme in which the C terminus of each MoaD subunit is deeply inserted into a MoaE subunit to form the active site. In the activated form of the enzyme, the MoaD C terminus is present as a thiocarboxylate. The present study identified the position of the thiocarboxylate sulfur by exploiting the anomalous signal originating from the sulfur atom. The structure of molybdopterin synthase in a novel crystal form revealed a binding pocket for the terminal phosphate of molybdopterin, the product of the enzyme, and suggested a binding site for the pterin moiety present in precursor Z and molybdopterin. Finally, the crystal structure of the MoaE homodimer provides insights into the conformational changes accompanying binding of the MoaD subunit.     

Mechanistic studies of human molybdopterin synthase reaction and characterization of mutants identified in group B patients of molybdenum cofactor deficiency

Biosynthesis of the molybdenum cofactor involves the initial formation of precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT. The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase. Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively. MOCS2A and MOCS2B were purified after heterologous expression in E. coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer. The rate of conversion of precursor Z to MPT by the human enzyme is slower than that of the eubacterial homologue. To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated. Characterization of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of precursor Z.     

Ubiquitin-Like Protein SAMP1 and JAMM/MPN+ Metalloprotease HvJAMM1 Constitute a System for Reversible Regulation of Metabolic Enzyme Activity in Archaea

Ubiquitin/ubiquitin-like (Ub/Ubl) proteins are involved in diverse cellular processes by their covalent linkage to protein substrates. Here, we provide evidence for a post-translational modification system that regulates enzyme activity which is composed of an archaeal Ubl protein (SAMP1) and a JAMM/MPN+ metalloprotease (HvJAMM1). Molybdopterin (MPT) synthase activity was found to be inhibited by covalent linkage of SAMP1 to the large subunit (MoaE) of MPT synthase. HvJAMM1 was shown to cleave the covalently linked inactive form of SAMP1-MoaE to the free functional individual SAMP1 and MoaE subunits of MPT synthase, suggesting reactivation of MPT synthase by this metalloprotease. Overall, this study provides new insight into the broad idea that Ub/Ubl modification is a post-translational process that can directly and reversibly regulate the activity of metabolic enzymes. In particular, we show that Ub/Ubl linkages on the active site residues of an enzyme (MPT synthase) can inhibit its catalytic activity and that the enzyme can be reactivated through cleavage by a JAMM/MPN+ metalloprotease.     

Role of the C-terminal Gly-Gly motif of Escherichia coli MoaD, a molybdenum cofactor biosynthesis protein with a ubiquitin fold

In Escherichia coli, the MoaD protein plays a central role in the conversion of precursor Z to molybdopterin (MPT) during molybdenum cofactor biosynthesis. MoaD has a fold similar to that of ubiquitin and contains a highly conserved C-terminal Gly-Gly motif, which in its active form contains a transferrable sulfur in the form of a thiocarboxylate group. During MPT biosynthesis, MoaD cycles between two different heterotetrameric complexes, one with MoaE to form MPT synthase and the other with MoeB, a protein similar to E1 in the ubiquitin pathway, to regenerate its transferrable sulfur. To determine the specific roles of each of the two terminal Gly residues with regard to the MoaD cycle, variants at the penultimate (Gly80) or terminal (Gly81) residues of both MoaD and thiocarboxylated MoaD were created. These variants were analyzed to determine their effects on complex formation with MoaE and MoeB, formation of the MoaD-acyl-adenylate complex, transfer of sulfur to precursor Z to form MPT, and total cofactor biosynthesis. The combined results show that while conservative substitutions at Gly80 had little effect on any of the processes that were examined, the terminal MoaD residue (Gly81) is important for transfer of sulfur to precursor Z and essential for formation of the MoaD-AMP complex. These results further our understanding of the mechanistic similarities of the MoaD-MoeB reaction to that of the ubiquitin-E1 system.     

Mechanistic and mutational studies of Escherichia coli molybdopterin synthase clarify the final step of molybdopterin biosynthesis

Biosynthesis of the molybdenum cofactor, a chelate of molybdenum or tungsten with a novel pterin, occurs in virtually all organisms including humans. In the cofactor, the metal is complexed to the unique cis-dithiolene moiety located on the pyran ring of molybdopterin. Escherichia coli molybdopterin synthase, the protein responsible for adding the dithiolene to a desulfo precursor termed precursor Z, is a dimer of dimers containing the MoaD and MoaE proteins. The sulfur used for dithiolene formation is carried in the form of a thiocarboxylate at the MoaD C terminus. Using an intein expression system for preparation of thiocarboxylated MoaD, the mechanism of the molybdopterin synthase reaction was examined. A stoichiometry of 2 molecules of thiocarboxylated MoaD per conversion of a single precursor Z molecule to molybdopterin was observed. Examination of several synthase variants bearing mutations in the MoaE subunit identified Lys-119 as a residue essential for activity and Arg-39 and Lys-126 as other residues critical for the reaction. An intermediate of the synthase reaction was identified and characterized. This intermediate remains tightly associated with the protein and is the predominant product formed by synthase containing the K126A variant of MoaE. Mass spectral data obtained from protein-bound intermediate are consistent with a monosulfurated structure that contains a terminal phosphate group similar to that present in molybdopterin.     

IscS Functions as a Primary Sulfur-donating Enzyme by Interacting Specifically with MoeB and MoaD in the Biosynthesis of Molybdopterin in Escherichia coli

The persulfide sulfur formed on an active site cysteine residue of pyridoxal 5′-phosphate-dependent cysteine desulfurases is subsequently incorporated into the biosynthetic pathways of a variety of sulfur-containing cofactors and thionucleosides. In molybdenum cofactor biosynthesis, MoeB activates the C terminus of the MoaD subunit of molybdopterin (MPT) synthase to form MoaD-adenylate, which is subsequently converted to a thiocarboxylate for the generation of the dithiolene group of MPT. It has been shown that three cysteine desulfurases (CsdA, SufS, and IscS) of Escherichia coli can transfer sulfur from l-cysteine to the thiocarboxylate of MoaD in vitro. Here, we demonstrate by surface plasmon resonance analyses that IscS, but not CsdA or SufS, interacts with MoeB and MoaD. MoeB and MoaD can stimulate the IscS activity up to 1.6-fold. Analysis of the sulfuration level of MoaD isolated from strains defective in cysteine desulfurases shows a largely decreased sulfuration level of the protein in an iscS deletion strain but not in a csdA/sufS deletion strain. We also show that another iscS deletion strain of E. coli accumulates compound Z, a direct oxidation product of the immediate precursor of MPT, to the same extent as an MPT synthase-deficient strain. In contrast, analysis of the content of compound Z in ΔcsdA and ΔsufS strains revealed no such accumulation. These findings indicate that IscS is the primary physiological sulfur-donating enzyme for the generation of the thiocarboxylate of MPT synthase in MPT biosynthesis.     

Equilibrium studies of a fluorescent paclitaxel derivative binding to microtubules

A fluorescent derivative of paclitaxel, 3'-N-m-aminobenzamido-3'-N-debenzamidopaclitaxel (N-AB-PT), has been prepared in order to probe paclitaxel-microtubule interactions. Fluorescence spectroscopy was used to quantitatively assess the association of N-AB-PT with microtubules. N-AB-PT was found equipotent with paclitaxel in promoting microtubule polymerization. Paclitaxel and N-AB-PT underwent rapid exchange with each other on microtubules assembled from GTP-, GDP-, and GMPCPP-tubulin. The equilibrium binding parameters for N-AB-PT to microtubules assembled from GTP-tubulin were derived through fluorescence titration. N-AB-PT bound to two types of sites on microtubules (K(d1) = 61 +/- 7.0 nM and K(d2) = 3.3 +/- 0.54 microM). The stoichiometry of each site was less than one ligand per tubulin dimer in the microtubule (n(1) = 0.81 +/- 0.03 and n(2) = 0.44 +/- 0.02). The binding experiments were repeated after exchanging the GTP for GDP or for GMPCPP. It was found that N-AB-PT bound to a single site on microtubules assembled from GDP-tubulin with a dissociation constant of 2.5 +/- 0.29 microM, and that N-AB-PT bound to a single site on microtubules assembled from GMPCPP-tubulin with a dissociation constant of 15 +/- 4.0 nM. It therefore appears that microtubules contain two types of binding sites for paclitaxel and that the binding site affinity for paclitaxel depends on the nucleotide content of tubulin. It has been established that paclitaxel binding does not inhibit GTP hydrolysis and microtubules assembled from GTP-tubulin in the presence of paclitaxel contain almost exclusively GDP at the E-site. We propose that although all the subunits of the microtubule at steady state are the same "GDP-tubulin-paclitaxel", they are formed through two paths: paclitaxel binding to a tubulin subunit before its E-site GTP hydrolysis is of high affinity, and paclitaxel binding to a tubulin subunit containing hydrolyzed GDP at its E-site is of low affinity.     

The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli

In the second step of the molybdenum cofactor (Moco) biosynthesis in Escherichia coli, the l-cysteine desulfurase IscS was identified as the primary sulfur donor for the formation of the thiocarboxylate on the small subunit (MoaD) of MPT synthase, which catalyzes the conversion of cyclic pyranopterin monophosphate to molybdopterin (MPT). Although in Moco biosynthesis in humans, the thiocarboxylation of the corresponding MoaD homolog involves two sulfurtransferases, an l-cysteine desulfurase, and a rhodanese-like protein, the rhodanese-like protein in E. coli remained enigmatic so far. Using a reverse approach, we identified a so far unknown sulfurtransferase for the MoeB-MoaD complex by protein-protein interactions. We show that YnjE, a three-domain rhodanese-like protein from E. coli, interacts with MoeB possibly for sulfur transfer to MoaD. The E. coli IscS protein was shown to specifically interact with YnjE for the formation of the persulfide group on YnjE. In a defined in vitro system consisting of MPT synthase, MoeB, Mg-ATP, IscS, and l-cysteine, YnjE was shown to enhance the rate of the conversion of added cyclic pyranopterin monophosphate to MPT. However, YnjE was not an enhancer of the cysteine desulfurase activity of IscS. This is the first report identifying the rhodanese-like protein YnjE as being involved in Moco biosynthesis in E. coli. We believe that the role of YnjE is to make the sulfur transfer from IscS for Moco biosynthesis more specific because IscS is involved in a variety of different sulfur transfer reactions in the cell.     

Human mitochondrial DNA polymerase holoenzyme: reconstitution and characterization

We have reconstituted the holoenzyme of the human mitochondrial DNA polymerase from cloned and overexpressed catalytic and accessory subunits. We have examined the polymerization activity of the catalytic subunit alone and of the holoenzyme to establish the function of the accessory subunit in this two subunit enzyme. The accessory subunit associates with the catalytic subunit with a dissociation constant of 35 +/- 16 nM as measured by the concentration dependence of its effect in stimulating maximal DNA binding and polymerization. At saturating concentrations, the accessory subunit contributes to every kinetic parameter examined to facilitate tighter binding of DNA and nucleotide and faster replication. The accessory protein makes the DNA binding 3.5-fold tighter (K(d) of 9.9 +/- 2.1 nM compared to 39 +/- 10 nM for the catalytic subunit alone) without significantly affecting the DNA dissociation rate (0.02 +/- 0.001 compared to 0.03 +/- 0.001 s(-)(1)). The ground-state nucleotide binding is improved from 4.7 +/- 2.0 to 0.78 +/- 0.065 microM, and the maximum DNA polymerization rate is increased from 8.7 +/- 1.1 to 45 +/- 1 s(-)(1) by the addition of the accessory protein. This leads to an increase in processivity from an estimated 290 +/- 46 to 2250 +/- 162. Although the accessory protein has been described as a "processivity factor" because of its effect on the ratio of rate constants defining processivity, this terminology falls short of adequately describing the profound effects of the small subunit on nucleotide-binding and incorporation catalyzed by the large subunit. By using the complete holoenzyme, we can now proceed with a comprehensive analysis of the structural and mechanistic determinants of enzyme specificity that govern toxicity of nucleoside analogues used in the treatment of viral infections such as AIDS.     

Cyanide binding to canine myeloperoxidase

Equilibria and kinetics of cyanide binding to canine myeloperoxidase were studied. Spectral results support the presence of two heme binding sites; an isosbestic point at 444 nm and a linear Scatchard plot suggest that the binding affinity of cyanide to the two subunits of the enzyme is the same. The dissociation constant is 0.53 microM. The pH dependence of the apparent second order rate constant indicates the presence of an acid-base group on the enzyme with a pKa of 3.8 +/- 0.1. The protonated form of cyanide binds to the basic enzyme with a rate constant of (4.3 +/- 0.3) x 10(6) M-1 s-1.     

Evidence for intersubunit communication during acetyl-CoA cleavage by the multienzyme CO dehydrogenase/acetyl-CoA synthase complex from Methanosarcina thermophila. Evidence that the beta subunit catalyzes C-C and C-S bond cleavage

The carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Methanosarcina thermophila is part of a five-subunit complex consisting of alpha, beta, gamma, delta, and epsilon subunits. The multienzyme complex catalyzes the reversible oxidation of CO to CO(2), transfer of the methyl group of acetyl-CoA to tetrahydromethanopterin (H(4)MPT), and acetyl-CoA synthesis from CO, CoA, and methyl-H(4)MPT. The alpha and epsilon subunits are required for CO oxidation. The gamma and delta subunits constitute a corrinoid iron-sulfur protein that is involved in the transmethylation reaction. This work focuses on the beta subunit. The isolated beta subunit contains significant amounts of nickel. When proteases truncate the beta subunit, causing the CODH/ACS complex to dissociate, the amount of intact beta subunit correlates directly with the EPR signal intensity of Cluster A and the activity of the CO/acetyl-CoA exchange reaction. Our results strongly indicate that the beta subunit harbors Cluster A, a NiFeS cluster, that is the active site of acetyl-CoA cleavage and assembly. Although the beta subunit is necessary, it is not sufficient for acetyl-CoA synthesis; interactions between the CODH and the ACS subunits are required for cleavage or synthesis of the C-C bond of acetyl-CoA. We propose that these interactions include intramolecular electron transfer reactions between the CODH and ACS subunits.     

Interaction of ADP and ATP with noncatalytic sites of isolated and membrane-bound chloroplast coupling factor CF<Subscript>1</Subscript>

This study of ATP and ADP binding to noncatalytic sites of membrane-bound CF1 (ATP synthase) revealed two noncatalytic sites with different specificities and affinities for nucleotides. One of these is characterized by a high affinity and specificity to ADP (Kd=2.6+/-0.3 microM). However, a certain increase in ADP apparent dissociation constant at high ATP/ADP ratio in the medium allows a possibility that ATP binds to this site as well. The other site displays high specificity to ATP. When the ADP-binding site is vacant, it shows a comparatively low affinity for ATP, which greatly increases with increasing ADP concentration accompanied by filling of the ADP-binding site. The reported specificities of these two sites are independent of thylakoid membrane energization, since both in the dark and in the light the ratios of ATP/ADP tightly bound to the noncatalytic sites were very close. The difference in noncatalytic site affinity for ATP and ADP is shown to depend on the amount of delta subunit in a particular sample. Thylakoid membrane ATP synthase, with stoichiometric content of delta-subunit (one delta-subunit per CF1 molecule), showed the maximal difference in ADP and ATP affinities for the noncatalytic sites. For CF1, with substoichiometric delta subunit values, this difference was less, and after delta subunit removal it decreased still more.     

Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation

Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea and eukaryotes, including humans. Genetic deficiencies of enzymes involved in Moco biosynthesis in humans lead to a severe and usually fatal disease. Moco contains a tricyclic pyranopterin, termed molybdopterin (MPT), that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of MPT is generated by MPT synthase, which consists of a large and small subunits. The 1.45 A resolution crystal structure of MPT synthase reveals a heterotetrameric protein in which the C-terminus of each small subunit is inserted into a large subunit to form the active site. In the activated form of the enzyme this C-terminus is present as a thiocarboxylate. In the structure of a covalent complex of MPT synthase, an isopeptide bond is present between the C-terminus of the small subunit and a Lys side chain in the large subunit. The strong structural similarity between the small subunit of MPT synthase and ubiquitin provides evidence for the evolutionary antecedence of the Moco biosynthetic pathway to the ubiquitin dependent protein degradation pathway.