Escherichia coli MoeA and MogA

Abstract

Escherichia coli MoeA and MogA are required for molybdenum cofactor biosynthesis and are believed to function in the addition of molybdenum to the dithiolene of molybdopterin to form molybdenum cofactor. Here we show thatmoeA − and mogA − cells are able to synthesize molybdopterin, but both are deficient in molybdenum incorporation and, as a consequence, are deficient in the formation of molybdopterin-guanine dinucleotide. Human sulfite oxidase expressed in E. coli moeA − could be activated in vitro in the presence of MoeA and low concentrations of molybdate. Sulfite oxidase purified from themoeA − lysate was also activated, although to a lesser extent than observed in the presence of lysate. MogA was incapable of activating sulfite oxidase expressed in E. coli mogA −. These results demonstrate that molybdenum insertion into molybdopterin is required for molybdopterin-guanine dinucleotide formation, and that MoeA facilitates molybdenum incorporation at low levels of molybdate, but MogA has an alternative function, possibly as a carrier for molybdopterin during molybdenum incorporation. Escherichia coli MoeA and MogA are required for molybdenum cofactor biosynthesis and are believed to function in the addition of molybdenum to the dithiolene of molybdopterin to form molybdenum cofactor. Here we show thatmoeA − and mogA − cells are able to synthesize molybdopterin, but both are deficient in molybdenum incorporation and, as a consequence, are deficient in the formation of molybdopterin-guanine dinucleotide. Human sulfite oxidase expressed in E. coli moeA − could be activated in vitro in the presence of MoeA and low concentrations of molybdate. Sulfite oxidase purified from themoeA − lysate was also activated, although to a lesser extent than observed in the presence of lysate. MogA was incapable of activating sulfite oxidase expressed in E. coli mogA −. These results demonstrate that molybdenum insertion into molybdopterin is required for molybdopterin-guanine dinucleotide formation, and that MoeA facilitates molybdenum incorporation at low levels of molybdate, but MogA has an alternative function, possibly as a carrier for molybdopterin during molybdenum incorporation. molybdenum cofactor molybdopterin molybdopterin guanine dinucleotide sulfite oxidase His-tagged SO dimethyl sulfoxide reductase His-tagged DMSOR high pressure liquid chromatography cell lysate containing expressed His-SO (SO)lysate depleted of His-SO nickel-nitrilotriacetic acid In Escherichia coli, the initial step in the molybdenum cofactor (Moco)1 biosynthetic pathway requires the moaA and moaC gene products to convert a guanosine nucleotide into precursor Z (1Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1995; 270: 1082-1087Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 2Reider C. Eisenreich W. O'Brien J. Richter G. Götze E. Boyle P. Blanchard S. Bacher A. Simon H. Eur. J. Biochem. 1998; 255: 24-36Crossref PubMed Scopus (69) Google Scholar). Molybdopterin (MPT) is synthesized from precursor Z by the products of the moaD, moaE, and moeB genes (3Pitterle D.M. Johnson J.L. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13506-13509Abstract Full Text PDF PubMed Google Scholar, 4Leimkühler S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 1837-1844Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 5Leimkühler S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 22024-22031Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 6Leimkühler S. Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 34695-34701Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and incorporation of molybdate into the MPT dithiolene yields Moco. InE. coli, the pterin is further modified to MPT-guanine dinucleotide (MGD) by the mobA gene product (7Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar, 8Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The mechanism of molybdenum incorporation remains one of the more enigmatic aspects of Moco biosynthesis. It is known that themoeA, mogA, and modABC gene products are all required for this step. While the ModA-C proteins comprise the molybdate transport system (9Grunden A.M. Shanmugam K.T. Arch. Microbiol. 1997; 168: 345-354Crossref PubMed Scopus (131) Google Scholar), the roles of MoeA and MogA have not been clearly delineated. Based on data showing partial recovery of nitrate reductase activity in an E. coli mogA−strain upon growth in high concentrations of Na2MoO4 (10Stewart V. MacGregor C.H. J. Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar) and the ability of purified MogA to bind MPT (11Liu M.T.W. Wuebbens M.M. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: 1814-1822Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), it has been proposed that MogA serves as a “molybdochelatase” (12Joshi M.S. Johnson J.L. Rajagopalan K.V. J. Bacteriol. 1996; 178: 4310-4312Crossref PubMed Google Scholar). A recent finding that xanthine dehydrogenase activity in a Rhodobacter capsulatus moeA − mutant could be recovered upon growth with 1 mm Na2MoO4 indicated that MoeA is also involved in molybdenum incorporation (13Leimkühler S. Angermüller S. Schwarz G. Mendel R.R. Klipp W. J. Bacteriol. 1999; 181: 5930-5939Crossref PubMed Google Scholar). Results from our laboratory have confirmed that E. coli MoeA also binds MPT. 2J. Nichols and K. V. Rajagopalan, unpublished results. Earlier studies from this laboratory have shown that fully functional human sulfite oxidase (SO) can be expressed in E. coli at high levels (14Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar). The data presented here show that SO expressed inmoeA − and mogA − cells contains normal levels of MPT, but is inactive due to a lack of molybdenum. This finding shows conclusively that bothmoeA − and mogA − cells synthesize MPT but are deficient in molybdenum incorporation. Additionally, MGD formation was found to be significantly reduced in these cell lines, indicating that molybdenum insertion is a prerequisite for nucleotide addition. An in vitro assay for MoeA function that monitors the MoO42−-dependent activation of heterologously expressed human SO in moeA −lysates is detailed, and conditions for optimal reconstitution are established. Parallel studies evaluating the ability of MogA to activate SO in mogA − lysates revealed that MogA was incapable of reconstituting SO activity, suggesting that the role of MogA in E. coli is distinct from that of MoeA. E. colistrains and plasmids used are listed in TableI.Table IE. coli strains and plasmidsGenotypeRef./SourceStrains BL21(DE3)F− ompThsdSB (r B−m B−)gal dcm (DE3)Novagen BW545 (moeA +)Δ(lacU) 169rpsL22Hasona A. Ray R.M. Shanmugam K.T. J. Bacteriol. 1998; 180: 1466-1472Crossref PubMed Google Scholar AH69 (moeA −) moeA − derivative of BW545 with kanamycin insert22Hasona A. Ray R.M. Shanmugam K.T. J. Bacteriol. 1998; 180: 1466-1472Crossref PubMed Google Scholar RK4353 (mogA +)ΔlacU169 araD139 rpsL gyrA non10Stewart V. MacGregor C.H. J. Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar RK5231 (mogA −) mogA − derivative of RK4353 with Mu insertion10Stewart V. MacGregor C.H. J. Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar moaA − moaA − K-12 strain formerly labeled chlA123Johnson M.E. Rajagopalan K.V. J. Bacteriol. 1987; 169: 117-125Crossref PubMed Google Scholar TP1000 (mobA −) mobAB − derivative of MC410024Palmer T. Santini C. Iobbi-Nivol C. Eaves D.J. Boxer D.H. Giordano G. Mol. Microbiol. 1996; 20: 875-884Crossref PubMed Scopus (146) Google ScholarPlasmids pJNeA11Expression vector forE. coli MoeA15Xiang S. Nichols J. Rajagopalan K.V. Schindelin H. Structure. 2001; 9: 299-310Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar pMW15gAExpression vector forE. coli MogA11Liu M.T.W. Wuebbens M.M. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: 1814-1822Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar pCT800AExpression vector forE. coli MobA 8Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar pTG718Expression vector for His-tagged human SO-K108R14Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar pTG918Expression vector for human SO-K108R14Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar pJH820Expression vector for His-taggedR. sphaeroides dimethyl sulfoxide reductase 8Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar Open table in a new tab E. coli MoeA and MogA were expressed and purified from BL21(DE3) as described previously (11Liu M.T.W. Wuebbens M.M. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: 1814-1822Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 15Xiang S. Nichols J. Rajagopalan K.V. Schindelin H. Structure. 2001; 9: 299-310Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). His-tagged human SO (His-SO) was expressed inmoeA −, moeA +,mogA −, mogA +, andmoaA − cells containing pTG718 (Table I). Cells were grown at 37 °C (with the exception ofmogA − cells which were grown at 30 °C) aerobically overnight in a 5-ml culture of LB medium containing 100 μg/ml ampicillin. This culture was used to inoculate 1 liter of LB/ampicillin, and His-SO was expressed aerobically at 30 °C in the presence of 50 μmisopropyl-β-d-thiogalactopyranoside for ∼19 h. Cells were grown without supplemental Na2MoO4unless otherwise indicated. The cultures were harvested by centrifugation and washed once with water. The cell paste was resuspended in 50 mm sodium phosphate, 300 mmNaCl, pH 8.0 (PN buffer) and lysed by three passages through a Microfluidics M110L Microfluidizer Processor. Lysates were centrifuged at 20,000 × g for 20 min. His-SO was purified in one step using a Ni-NTA-agarose affinity column (Qiagen) as described by Temple et al. (14Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar), and the purified SO was dialyzed into 50 mm Tris, pH 7.5. The protein was quantitated spectrophotometrically at 413 nm using an extinction coefficient of 113,000 m−1 cm−1 (2.174 (mg/ml)−1 cm−1) (16Rajagopalan K.V. Coughlan M.P. Molybdenum and Molybdenum-Containing Enzymes. Pergamon, Oxford1980: 241-272Google Scholar). The activity of SO was assayed by monitoring the reduction of cytochrome c at 550 nm as described previously (17Garrett R.M. Rajagopalan K.V. J. Biol. Chem. 1996; 271: 7387-7391Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). One unit of SO activity is defined as an absorbance change of 1.0/min at 25 °C. His-tagged dimethyl sulfoxide reductase (His-DMSOR) was expressed inmoeA −, moeA +,mogA −, mogA +, andmobA − cells containing pJH820 (Table I) as described by Temple et al. (8Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Isopropyl-β-d-thiogalactopyranoside was used at 10 μm for induction, and no supplemental Na2MoO4 was added to the cultures unless otherwise indicated. His-DMSOR was purified using the same procedure as for His-SO except that the enzyme was eluted from the resin with PN buffer containing 100 mm imidazole instead of 200 mm. His-DMSOR was quantitated using an extinction coefficient of 200,000 m−1 cm−1(2.3 (mg/ml)−1 cm−1) at 280 nm (18Hilton J.C. Rajagopalan K.V. Arch. Biochem. Biophys. 1996; 325: 139-143Crossref PubMed Scopus (46) Google Scholar). DMSOR activity was measured as described previously (19Hilton J.C. Temple C.A. Rajagopalan K.V. J. Biol. Chem. 1999; 274: 8428-8436Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), with one unit of activity defined as 1.0 μmol of Me2SO reduced per min at 25 °C. The MPT content of His-SO was quantitated by conversion to the fluorescent Form A (dephospho) derivative (20Johnson J.L. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6856-6860Crossref PubMed Scopus (273) Google Scholar) using 100 μg of His-SO in a total volume of 800 μl of 50 mm Tris, pH 7.5. The presence of dephospho-Form A was monitored using an Agilent 1100 Series fluorescence detector with an excitation wavelength of 295 nm and emission at 448 nm. The MGD content of His-DMSOR was assayed by quantitation of Form by A of purified DMSOR in 50 mm Tris, pH was to pH and with 50 μl of at in the for h. The was with and was a column with mm The of the was overnight at with unit of nucleotide and μl of and the same of Form Form was quantitated by the in Form A fluorescence Form A is more fluorescent than Form Molybdenum content was as described previously (19Hilton J.C. Temple C.A. Rajagopalan K.V. J. Biol. Chem. 1999; 274: 8428-8436Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) using a MPT and MGD levels were assayed by the of Form A and Form from cells without a were resuspended in of of lysed by passages through a pressure cell at and centrifuged for 20 min at 20,000 × The was as described previously (7Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar) and to a with 50 of and A were eluted using of mm and Form was eluted with of 50 mm containing and A were to 100 mm Tris, 100 pH and of overnight the was to a The column was washed with 30 of and dephospho-Form A was eluted with of 10 mm containing Form A were and quantitated as described (7Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar). from the initial column containing Form were with and to in 1 of the was a column with mm and 30 was and resuspended in 1 of 100 mm Tris, 100 mm pH The resuspended was and was as described for quantitation of MGD in purified SO and were expressed and purified from mobA − − containing as described (8Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) except that was expressed in the of supplemental MPT was from SO by for 1 min at 100 °C. MobA and were expressed and purified − cells as described previously (8Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). of MGD and activated DMSOR was monitored as described (8Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) except that no was His-SO was expressed in cultures of moeA − − cells as described without supplemental the cells were resuspended in 10 of 50 mm Tris, pH with 1 μg/ml 1 mm and 10 μg/ml Cells were lysed by passages through a pressure cell at and the lysate was centrifuged at 20,000 for 20 min. The labeled was in at °C. The of SO in the lysate was spectrophotometrically as the of SO purified from a using a was in 50 mm Tris, pH 7.5. MoeA MogA and Na2MoO4 in a total of 50 μl were added to 50 μl of at 37 °C for assayed for SO His-SO was purified from of (moeA −) and (mogA −) by lysates Ni-NTA-agarose with 50 mm Tris, pH 7.5. The labeled were the column with of PN buffer containing 20 mm the SO was eluted with of PN buffer containing 200 mm The SO was into 50 mm Tris, pH using a column and to μl using a 30 concentrations were using the assay with as a studies were as described in the and for lysate protein It has previously been that growth at high concentrations of Na2MoO4 activate nitrate reductase in E. coli mogA − cells (10Stewart V. MacGregor C.H. J. Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar) and xanthine dehydrogenase capsulatus moeA − cells (13Leimkühler S. Angermüller S. Schwarz G. Mendel R.R. Klipp W. J. Bacteriol. 1999; 181: 5930-5939Crossref PubMed Google Scholar). In the of His-SO and His-DMSOR expressed inmoeA − and mogA − E. coli cells were not by the of Na2MoO4 in the growth at high the of both were and molybdenum content was of that of proteins purified from cells not of SO and DMSOR from moeA − and mogA − in growth was assayed by monitoring reduction of cytochrome was assayed by monitoring reduction of was assayed by monitoring reduction of cytochrome was assayed by monitoring reduction of Open table in a new tab of the MPT content of His-SO purified from the mutant strains revealed that both the moeA − − strains His-SO containing MPT in the of in the growth His-SO purified from − which has a in the step of cofactor was of MPT. The of Form A from mogA − His-SO was found to be than that from the of Form A from SO to of the MPT content J.L. Rajagopalan K.V. J. Biol. Chem. Full Text PDF PubMed Google Scholar), it is that the of of MPT to SO expressed in − strain yields levels of Form A. of MGD in His-DMSOR purified from the mutant strains a of the dinucleotide form of the of the in the strain content of His-DMSOR from moeA − and mogA − was to the Form derivative for Form are as the in Form A fluorescence addition of per of of MGD was to the Form derivative for Form are as the in Form A fluorescence addition of per of Open table in a new tab the that moeA − − cells can form MGD but it to MPT and MGD levels in themoeA − and mogA − cell strains were quantitated and with of in all strains levels of MPT. themoeA − the mogA levels of the of MGD in the His-DMSOR purified from these and MGD levels in cell was to Form A for the fluorescence of Form is the of at three was to Form for for Form the total fluorescence of Form A addition of MPT was to Form A for the fluorescence of Form is the of at three MGD was to Form for for Form the total fluorescence of Form A addition of Open table in a new tab The of molybdenum insertion to conversion of MPT to MGD was further by monitoring the ability of purified E. coli MobA to convert MPT and to MPT and were by of SO from themoeA − − (8Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The no activity for and but an MPT content to that of The results in from is a for MobA and is to the MPT from is Additionally, it was observed that MPT from is as an MPT for the activation of in the presence of 10 mm Na2MoO4 not have shown that MPT synthesized in vitro system S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 1837-1844Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) can be into SO to enzyme in the presence of high concentrations of MoeA can molybdenum incorporation into MPT at molybdenum an in vitro system was to the reconstitution of SO in − cell lysate. His-SO was expressed inmoeA − cells without Na2MoO4 and were as described and In the of low levels of SO activity were observed reconstitution was in the presence of MoeA at low concentrations of molybdate. The activity in the presence of MoeA was of that of SO expressed inmoeA cells with 1 (Table The addition of MoeA no on SO activity in the of of MoeA were added to containing 10 activation was at μm MoeA with no of activation at MoeA levels as high as was at 37 with the extent in min MogA could also MoO42−-dependent activation of His-SO was expressed in the mogA − cell strain and the cell lysate as for the was assayed for activation with of Na2MoO4 and no in activation was observed shown in MogA was also incapable of activating SO expressed in moeA − and MoeA was to SO activation in The addition of both MogA and MoeA to no on the of SO activation observed with MoeA While MoeA was able to activate the presence of the mogA it could not activate in the presence of and MogA was to activate SO The partial activation of the of and observed upon addition of MogA can be to the presence of MoeA in the the addition of buffer results in the same extent of function of MogA is required to activation of SO by MoeA. 10 the of 50 μl of lysate at the of purified SO to μm and μm MoeA μm MogA μm MoeA μm MogA buffer were for min at 37 of purified by molybdate and MoeA in the presence and of lysate was and the results are presented in of molybdenum in activation of the purified not μm MoeA and 10 purified was activated to of the in molybdenum concentrations to 10 mm in activation to of that with not was added to the the extent of activation was the same as was with the (Table using purified moaA −) in of and expressed in of no activation not addition of could activation with that of SO in the lysate is not activation by MoeA requires the protein of addition to the listed lysate and SO all 10 and μm MoeA in a volume with 50 mm Tris, pH 7.5. SO was at and 50 μl of lysate buffer was used the protein of μl was of activity observed for in containing lysate were to for the lysate protein in the protein In addition to the listed lysate and SO all 10 and μm MoeA in a volume with 50 mm Tris, pH 7.5. SO was at and 50 μl of lysate buffer was used the protein of μl was in containing lysate were to for the lysate protein in the Open table in a new tab the of lysate on SO activation was on the protein in the was on a of the from not ability to activate (Table indicating that the protein of the lysate was The described here were an of the roles of MoeA and MogA in the biosynthesis of MPT and MGD and molybdenum incorporation into Moco. this His-tagged human SO and Rhodobacter sphaeroides DMSOR were expressed and purified from moeA − − cells and for activity, molybdenum and MPT and MGD The data presented here conclusively show that in mogA have no on MPT the of molybdenum to MPT. both moeA − and mogA levels of MPT with of His-SO and His-DMSOR purified from cell strain are inactive due to the of molybdenum the cells are grown with high concentrations of − and mogA − cells are of the dinucleotide form of the cofactor required by In vitro of the formation of MGD that no MGD is SO purified − cells was used as a pterin These clearly molybdenum as a prerequisite for nucleotide addition. further the role of MoeA in Moco a procedure was that monitors the MoO42−-dependent activation of SO in by MoeA. of SO by MoeA and molybdate was the was in the lysate than with the could be activated to a but addition of in activation with that observed with the Results using indicated that the was for the suggesting that the by the lysate was to be a the of an to a protein in the high be studies are at the and of the required for Based on the that nitrate reductase activity − cells can be by growth on high concentrations of Na2MoO4 and the ability of MogA to bind MPT, it has been that MogA as a (10Stewart V. MacGregor C.H. J. Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar, M.T.W. Wuebbens M.M. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: 1814-1822Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, M.S. Johnson J.L. Rajagopalan K.V. J. Bacteriol. 1996; 178: 4310-4312Crossref PubMed Google Scholar). the of MogA to the MoO42−-dependent activation of SO expressed in mogA − cells is not with this results have not defined a role for the data for MoeA and with MogA required in a step that the The presence of MogA also to be required for insertion of MPT into The that could not be significantly activated by molybdate containing MPT, that an in of the of MPT in activation by molybdate. The function of MogA be to bind MPT in the during the molybdenum insertion step in The ability of MogA to bind MPT to this (11Liu M.T.W. Wuebbens M.M. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: 1814-1822Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). laboratory has in vitro for MPT and for MGD biosynthesis S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 1837-1844Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 8Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This the functional assay for the molybdenum insertion step of Moco biosynthesis and a the of MoeA and MogA in this step. In are to be by Moco than MPT, and MoeA function to MPT. The of a MoO42−-dependent assay for MoeA the for further into role in molybdenum are at the required for optimal activation of His-SO using a more The data from these also to more clearly the function of MogA in molybdenum