In Vitro Molybdenum Ligation to Molybdopterin Using Purified Components

Abstract

We have previously shown that Escherichia coli MoeA and MogA are required in vivo for the final step of molybdenum cofactor biosynthesis, the addition of the molybdenum atom to the dithiolene of molybdopterin. MoeA was also shown to facilitate the addition of molybdenum in an assay using crude extracts from E. coli moeA ؊ cells. The experiments detailed in this report utilized an in vitro assay for MoeA-mediated molybdenum ligation to de novo synthesized molybdopterin using only purified components and monitoring the reconstitution of human aposulfite oxidase. In this assay, maximum activation was achieved by delaying the addition of aposulfite oxidase to allow for adequate molybdenum coordination to occur. Tungsten, which substitutes for molybdenum in hyperthermophilic organisms, could also be ligated to molybdopterin using this system, though not as efficiently as molybdenum. Addition of thiol compounds to the assay inhibited activity. Addition of MogA also inhibited the reaction. However, in the presence of ATP and magnesium, addition of MogA to the assay increased the level of aposulfite oxidase reconstitution beyond that observed with MoeA alone. This effect was not observed in the absence of MoeA. The results presented here demonstrate that MoeA is responsible for mediating molybdenum ligation to molybdopterin, whereas MogA stimulates this activity in an ATP-dependent manner.In all molybdenum-containing enzymes, with the exception of nitrogenase, the molybdenum cofactor (Moco) 1 consists of a mononuclear Mo atom coordinated to the cis-dithiolene moiety of molybdopterin (MPT). In Escherichia coli, biosynthesis of Moco begins with the conversion of GTP to a pterin intermediate termed precursor Z, catalyzed by the MoaA and MoaC proteins (1-4). MPT is synthesized from precursor Z by the MoaD and MoaE proteins, which together compose the MPT synthase complex (5, 6). The MoeB protein is involved in reactivating the MoaD subunit of MPT synthase between rounds of MPT synthesis (7,8). Ligation of the Mo atom to MPT requires the MoeA and MogA proteins along with the ModABC molybdate transporter system (9, 10). In E. coli, the cofactor is further modified by the covalent addition of GMP to the MPT phosphate, a reaction catalyzed by the MobA protein (11,12).Previous results from our laboratory verified that E. coli MoeA and MogA are both required for in vivo Mo ligation to the MPT dithiolene. Analysis of crude extracts produced from E. coli moeA Ϫ and mogA Ϫ cells showed the presence of MPT but the absence of MPT-guanine dinucleotide, the biosynthesis of which was found to require prior attachment of Mo to MPT. Recombinantly expressed human sulfite oxidase (SO) purified from both strains contained MPT but was devoid of Mo. In vitro experiments demonstrated the ability of MoeA to activate the recombinant Mo-free, MPT-containing human SO in moeA Ϫ crude extract. MogA was incapable of supporting the same type of activity in mogA Ϫ crude extract. Thus, MoeA appeared to be the protein directly responsible for Mo ligation, ...

Identification of a Bis-molybdopterin Intermediate in Molybdenum Cofactor Biosynthesis in Escherichia coli

“…An MPT/molybdenum ratio close to 1:1 was found in hSO, consistent with the quantitative presence of the Mo-MPT cofactor in the enzyme. A similar ratio was determined for MoeA incubated with Mo-MPT, supporting previous suggestions that this protein is able to bind exogenously added Mo-MPT (42,43). These controls emphasized the accuracy of the used method for detection of the MPT/molybdenum ratio.…”

MocA Is a Specific Cytidylyltransferase Involved in Molybdopterin Cytosine Dinucleotide Biosynthesis in Escherichia coli

“…Moco biosynthesis has been extensively studied in Escherichia coli by using a combination of biochemical, genetic, and structural approaches (1,2). The biosynthesis of Moco has been divided into four major steps in Escherichia coli: (i) formation of precursor Z (3,4), (ii) formation of MPT from precursor Z (5, 6), (iii) insertion of molybdenum to form Moco via an adenylylated MPT intermediate (7)(8)(9), and (iv) additional modification by covalent addition of GMP to the C4Ј phosphate of MPT via a pyrophosphate bond, forming the molybdopterin guanine dinucleotide (MGD) cofactor (10,11). In E. coli, GMP attachment to Moco is catalyzed by the MobA and MobB proteins (12).…”

Mutational Analysis of Escherichia coli MoeA: Two Functional Activities Map to the Active Site Cleft,

“…The first two possibilities are unlikely as, in the absence of lysate, MoeA was only able to activate SO at ~5 % of the level observed in the presence of lysate (9). Furthermore, the premature addition of apo-SO to the purified component system lowers the efficiency of activation, and apo-SO binds MPT/Moco quite readily from reactions containing a 10-fold excess of MoeA (14). Therefore, activity in crude extract is likely to be dependent upon the ability of MoeA to interact with some unidentified cellular factor(s) in order to mediate Mo ligation.…”

“…Assays to monitor the ability of the MoeA variants to mediate the activation of apo-SO using purified components were performed by the method described for wild-type MoeA (14). Data was analyzed using Kaleidograph software (Synergy).…”

“…The development of the recently described fully defined assay for MoeA (14) provided the opportunity for a more direct analysis of the effects of amino acid substitutions in MoeA. The results from these experiments, shown in Fig.…”