Portal de Periódicos da CAPES

Methionine synthase is localized to the nucleus in Pichia pastoris and Candida albicans and to the cytoplasm in Saccharomyces cerevisiae

2017; Elsevier BV; Volume: 292; Issue: 36 Linguagem: Inglês

10.1074/jbc.m117.783019

1083-351X

Umakant Sahu, Vinod Rajendra, Shankar S. Kapnoor, Raghu Bhagavat, Nagasuma Chandra, Pundi N. Rangarajan,

Folate and B Vitamins Research

Methionine synthase (MS) 3The abbreviations used are: MS, methionine synthase; PpMS, MS encoded by P. pastoris genome; ScMS, MS encoded by S. cerevisiae genome; CaMS, MS encoded by C. albicans genome; MAT, methionine adenosyltransferase; THF, tetrahydrofolate; NES, nuclear export signal; MAS, membrane anchor signal; NLS, nuclear localization signal; TRITC, tetramethylrhodamine isothiocyanate; PDB, Protein Data Bank; F, forward; R, reverse. 3The abbreviations used are: MS, methionine synthase; PpMS, MS encoded by P. pastoris genome; ScMS, MS encoded by S. cerevisiae genome; CaMS, MS encoded by C. albicans genome; MAT, methionine adenosyltransferase; THF, tetrahydrofolate; NES, nuclear export signal; MAS, membrane anchor signal; NLS, nuclear localization signal; TRITC, tetramethylrhodamine isothiocyanate; PDB, Protein Data Bank; F, forward; R, reverse. catalyzes methylation of homocysteine, the last step in the biosynthesis of methionine, which is essential for the regeneration of tetrahydrofolate and biosynthesis of S-adenosylmethionine. Here, we report that MS is localized to the nucleus of Pichia pastoris and Candida albicans but is cytoplasmic in Saccharomyces cerevisiae. The P. pastoris strain carrying a deletion of the MET6 gene encoding MS (Ppmet6) exhibits methionine as well as adenine auxotrophy indicating that MS is required for methionine as well as adenine biosynthesis. Nuclear localization of P. pastoris MS (PpMS) was abrogated by the deletion of 107 C-terminal amino acids or the R742A mutation. In silico analysis of the PpMS structure indicated that PpMS may exist in a dimer-like configuration in which Arg-742 of a monomer forms a salt bridge with Asp-113 of another monomer. Biochemical studies indicate that R742A as well as D113R mutations abrogate nuclear localization of PpMS and its ability to reverse methionine auxotrophy of Ppmet6. Thus, association of two PpMS monomers through the interaction of Arg-742 and Asp-113 is essential for catalytic activity and nuclear localization. When PpMS is targeted to the cytoplasm employing a heterologous nuclear export signal, it is expressed at very low levels and is unable to reverse methionine and adenine auxotrophy of Ppmet6. Thus, nuclear localization is essential for the stability and function of MS in P. pastoris. We conclude that nuclear localization of MS is a unique feature of respiratory yeasts such as P. pastoris and C. albicans, and it may have novel moonlighting functions in the nucleus. Methionine synthase (MS) 3The abbreviations used are: MS, methionine synthase; PpMS, MS encoded by P. pastoris genome; ScMS, MS encoded by S. cerevisiae genome; CaMS, MS encoded by C. albicans genome; MAT, methionine adenosyltransferase; THF, tetrahydrofolate; NES, nuclear export signal; MAS, membrane anchor signal; NLS, nuclear localization signal; TRITC, tetramethylrhodamine isothiocyanate; PDB, Protein Data Bank; F, forward; R, reverse. 3The abbreviations used are: MS, methionine synthase; PpMS, MS encoded by P. pastoris genome; ScMS, MS encoded by S. cerevisiae genome; CaMS, MS encoded by C. albicans genome; MAT, methionine adenosyltransferase; THF, tetrahydrofolate; NES, nuclear export signal; MAS, membrane anchor signal; NLS, nuclear localization signal; TRITC, tetramethylrhodamine isothiocyanate; PDB, Protein Data Bank; F, forward; R, reverse. catalyzes methylation of homocysteine, the last step in the biosynthesis of methionine, which is essential for the regeneration of tetrahydrofolate and biosynthesis of S-adenosylmethionine. Here, we report that MS is localized to the nucleus of Pichia pastoris and Candida albicans but is cytoplasmic in Saccharomyces cerevisiae. The P. pastoris strain carrying a deletion of the MET6 gene encoding MS (Ppmet6) exhibits methionine as well as adenine auxotrophy indicating that MS is required for methionine as well as adenine biosynthesis. Nuclear localization of P. pastoris MS (PpMS) was abrogated by the deletion of 107 C-terminal amino acids or the R742A mutation. In silico analysis of the PpMS structure indicated that PpMS may exist in a dimer-like configuration in which Arg-742 of a monomer forms a salt bridge with Asp-113 of another monomer. Biochemical studies indicate that R742A as well as D113R mutations abrogate nuclear localization of PpMS and its ability to reverse methionine auxotrophy of Ppmet6. Thus, association of two PpMS monomers through the interaction of Arg-742 and Asp-113 is essential for catalytic activity and nuclear localization. When PpMS is targeted to the cytoplasm employing a heterologous nuclear export signal, it is expressed at very low levels and is unable to reverse methionine and adenine auxotrophy of Ppmet6. Thus, nuclear localization is essential for the stability and function of MS in P. pastoris. We conclude that nuclear localization of MS is a unique feature of respiratory yeasts such as P. pastoris and C. albicans, and it may have novel moonlighting functions in the nucleus. Methionine synthase (MS) 3The abbreviations used are: MS, methionine synthase; PpMS, MS encoded by P. pastoris genome; ScMS, MS encoded by S. cerevisiae genome; CaMS, MS encoded by C. albicans genome; MAT, methionine adenosyltransferase; THF, tetrahydrofolate; NES, nuclear export signal; MAS, membrane anchor signal; NLS, nuclear localization signal; TRITC, tetramethylrhodamine isothiocyanate; PDB, Protein Data Bank; F, forward; R, reverse. catalyzes the terminal step of de novo biosynthesis of methionine. It transfers a methyl group from 5-methyltetrahydrofolate to l-homocysteine to form l-methionine and tetrahydrofolate (THF) (1.González J.C. Banerjee R.V. Huang S. Sumner J.S. Matthews R.G. Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem.Biochemistry. 1992; 31: 6045-6056Crossref PubMed Scopus (120) Google Scholar). Two functionally and structurally distinct types of MS have been identified: cobalamin-dependent and cobalamin-independent enzymes of ∼140,000 and ∼86 kDa size, respectively (2.Matthews R.G. Smith A.E. Zhou Z.S. Taurog R.E. Bandarian V. Evans J.C. Ludwig M. Cobalamin-dependent and cobalamin-independent methionine synthases: are there two solutions to the same chemical problem?.Helv. Chim. Acta. 2003; 86: 3939-3954Crossref Scopus (51) Google Scholar). Although bacteria such as Escherichia coli contain both types of MS, mammals, including humans, have only the cobalamin-dependent enzyme. Fungi, plants, and some bacteria lack the ability to obtain cobalamin or to synthesize cobalamin de novo and therefore possess only the cobalamin-independent enzyme (2.Matthews R.G. Smith A.E. Zhou Z.S. Taurog R.E. Bandarian V. Evans J.C. Ludwig M. Cobalamin-dependent and cobalamin-independent methionine synthases: are there two solutions to the same chemical problem?.Helv. Chim. Acta. 2003; 86: 3939-3954Crossref Scopus (51) Google Scholar). Despite catalyzing similar reactions, the cobalamin-dependent and -independent enzymes exhibit significant differences in their structure as well as mechanism of action, and therefore, the latter are attractive targets for the development of antifungals against pathogenic fungi such as Candida albicans and Cryptococcus neoformans (3.Suliman H.S. Appling D.R. Robertus J.D. The gene for cobalamin-independent methionine synthase is essential in Candida albicans: a potential antifungal target.Arch. Biochem. Biophys. 2007; 467: 218-226Crossref PubMed Scopus (35) Google Scholar, 4.Pascon R.C. Ganous T.M. Kingsbury J.M. Cox G.M. McCusker J.H. Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence.Microbiology. 2004; 150: 3013-3023Crossref PubMed Scopus (61) Google Scholar). The crystal structures of cobalamin-independent MS of E. coli, Thermogota maritima, Arabidopsis thaliana, and C. albicans are known (5.González J.C. Peariso K. Penner-Hahn J.E. Matthews R.G. Cobalamin-independent methionine synthase from Escherichia coli: a zinc metalloenzyme.Biochemistry. 1996; 35: 12228-12234Crossref PubMed Scopus (126) Google Scholar6.Pejchal R. Ludwig M.L. Cobalamin-independent methionine synthase (MetE): a face-to-face double barrel that evolved by gene duplication.PLoS Biol. 2005; 3: e31Crossref PubMed Scopus (96) Google Scholar, 7.Ferrer J.L. Ravanel S. Robert M. Dumas R. Crystal structures of cobalamin-independent methionine synthase complexed with zinc, homocysteine, and methyltetrahydrofolate.J. Biol. Chem. 2004; 279: 44235-44238Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 8.Ubhi D. Kavanagh K.L. Monzingo A.F. Robertus J.D. Structure of Candida albicans methionine synthase determined by employing surface residue mutagenesis.Arch. Biochem. Biophys. 2011; 513: 19-26Crossref PubMed Scopus (6) Google Scholar9.Ubhi D. Kago G. Monzingo A.F. Robertus J.D. Structural analysis of a fungal methionine synthase with substrates and inhibitors.J. Mol. Biol. 2014; 426: 1839-1847Crossref PubMed Scopus (9) Google Scholar). In these species, MS was shown to fold into two (βα)8 barrels with the N- and C-terminal barrels joined by an inter-domain linker. C. albicans MS (CaMS) consists of two (βα)8 barrels, and the active site is located between the two domains (8.Ubhi D. Kavanagh K.L. Monzingo A.F. Robertus J.D. Structure of Candida albicans methionine synthase determined by employing surface residue mutagenesis.Arch. Biochem. Biophys. 2011; 513: 19-26Crossref PubMed Scopus (6) Google Scholar, 9.Ubhi D. Kago G. Monzingo A.F. Robertus J.D. Structural analysis of a fungal methionine synthase with substrates and inhibitors.J. Mol. Biol. 2014; 426: 1839-1847Crossref PubMed Scopus (9) Google Scholar). Binding sites for a zinc ion and the substrates l-homocysteine and 5-methyltetrahydrofolate glutamate have also been mapped. Binding of l-homocysteine or methionine was shown to result in conformational rearrangements at the amino acid binding pocket, moving the catalytic zinc into position to activate the thiol group (8.Ubhi D. Kavanagh K.L. Monzingo A.F. Robertus J.D. Structure of Candida albicans methionine synthase determined by employing surface residue mutagenesis.Arch. Biochem. Biophys. 2011; 513: 19-26Crossref PubMed Scopus (6) Google Scholar, 9.Ubhi D. Kago G. Monzingo A.F. Robertus J.D. Structural analysis of a fungal methionine synthase with substrates and inhibitors.J. Mol. Biol. 2014; 426: 1839-1847Crossref PubMed Scopus (9) Google Scholar). Yeast strains carrying a deletion in the gene encoding MS exhibit multiple phenotypes. In Saccharomyces cerevisiae and Aspergillus nidulans, methionine auxotrophy caused by MS dysfunction can be fully rescued by methionine supplementation (3.Suliman H.S. Appling D.R. Robertus J.D. The gene for cobalamin-independent methionine synthase is essential in Candida albicans: a potential antifungal target.Arch. Biochem. Biophys. 2007; 467: 218-226Crossref PubMed Scopus (35) Google Scholar, 10.Kacprzak M.M. Lewandowska I. Matthews R.G. Paszewski A. Transcriptional regulation of methionine synthase by homocysteine and choline in Aspergillus nidulans.Biochem. J. 2003; 376: 517-524Crossref PubMed Google Scholar). However, C. albicans carrying deletion of both the alleles encoding MS (met6/met6) cannot grow on media supplemented with exogenous methionine (3.Suliman H.S. Appling D.R. Robertus J.D. The gene for cobalamin-independent methionine synthase is essential in Candida albicans: a potential antifungal target.Arch. Biochem. Biophys. 2007; 467: 218-226Crossref PubMed Scopus (35) Google Scholar). Similarly, the met6 strain of C. neoformans grows at a slow rate in minimal media supplemented with methionine (4.Pascon R.C. Ganous T.M. Kingsbury J.M. Cox G.M. McCusker J.H. Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence.Microbiology. 2004; 150: 3013-3023Crossref PubMed Scopus (61) Google Scholar), whereas the met6 strain of Fusarium graminearum is defective in aerial hyphal growth when cultured on methionine-supplemented media (11.Seong K. Hou Z. Tracy M. Kistler H.C. Xu J.R. Random insertional mutagenesis identifies genes associated with virulence in the wheat scab fungus Fusarium graminearum.Phytopathology. 2005; 95: 744-750Crossref PubMed Scopus (132) Google Scholar). In the case of Schizosaccharomyces pombe, disruption of met26 encoding MS results in methionine as well as adenine auxotrophy due to homocysteine accumulation and defective purine biosynthesis (12.Fujita Y. Ukena E. Iefuji H. Giga-Hama Y. Takegawa K. Homocysteine accumulation causes a defect in purine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs.Microbiology. 2006; 152: 397-404Crossref PubMed Scopus (21) Google Scholar). Thus, in addition to its role in methionine biosynthesis, MS may have other unknown functions in certain yeast species. The genome of P. pastoris, a methylotrophic yeast, encodes a cobalamin-independent MS (PpMS), which shares 77 and 79% amino acid sequence identity with the MS of S. cerevisiae (ScMS) and CaMS, respectively (13.Huang L. Li D.Y. Wang S.X. Zhang S.M. Chen J.H. Wu X.F. Cloning and identification of methionine synthase gene from Pichia pastoris.Acta Biochim. Biophys. Sin. 2005; 37: 371-378Crossref Scopus (2) Google Scholar). Here, we report that deletion of MET6 results in methionine as well as adenine auxotrophy in P. pastoris but only methionine auxotrophy in S. cerevisiae. MS is uniquely localized to the nucleus of P. pastoris and C. albicans, although it localizes to the cytosol in S. cerevisiae. Arg-742 and Asp-113 located in the C- and N-terminal regions of PpMS interact with each other and facilitate association between two monomers in a dimer-like configuration. Interaction between Arg-742 and Asp-113 is essential for the stability, catalytic activity, and nuclear localization of MS. Taken together, our results suggest that MS is present in the nucleus of respiratory yeasts such as P. pastoris and C. albicans, and it possesses unique biochemical properties that have not been reported in any other species. Our laboratory has been studying key transcription factors involved in the regulation of carbon metabolism in the respiratory yeast, P. pastoris (14.Kranthi B.V. Balasubramanian N. Rangarajan P.N. Isolation of a single-stranded DNA-binding protein from the methylotrophic yeast, Pichia pastoris and its identification as ζ crystallin.Nucleic Acids Res. 2006; 34: 4060-4068Crossref PubMed Scopus (22) Google Scholar15.Kranthi B.V. Kumar R. Kumar N.V. Rao D.N. Rangarajan P.N. Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris.Biochim. Biophys. Acta. 2009; 1789: 460-468Crossref PubMed Scopus (40) Google Scholar, 16.Kranthi B.V. Kumar H.R. Rangarajan P.N. Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris.Yeast. 2010; 27: 705-711Crossref PubMed Scopus (27) Google Scholar, 17.Kumar N.V. Rangarajan P.N. Catabolite repression of phosphoenolpyruvate carboxykinase by a zinc finger protein under biotin- and pyruvate carboxylase-deficient conditions in Pichia pastoris.Microbiology. 2011; 157: 3361-3369Crossref PubMed Scopus (15) Google Scholar, 18.Kumar N.V. Rangarajan P.N. The zinc finger proteins Mxr1p and repressor of phosphoenolpyruvate carboxykinase (ROP) have the same DNA binding specificity but regulate methanol metabolism antagonistically in Pichia pastoris.J. Biol. Chem. 2012; 287: 34465-34473Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 19.Sahu U. Krishna Rao K. Rangarajan P.N. Trm1p, a Zn(II)(2)Cys(6)-type transcription factor, is essential for the transcriptional activation of genes of methanol utilization pathway, in Pichia pastoris.Biochem. Biophys. Res. Commun. 2014; 451: 158-164Crossref PubMed Scopus (38) Google Scholar, 20.Sahu U. Rangarajan P.N. Regulation of acetate metabolism and acetyl-CoA synthetase 1 (ACS1) expression by methanol expression regulator 1 (Mxr1p) in the methylotrophic yeast Pichia pastoris.J. Biol. Chem. 2016; 291: 3648-3657Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar21.Sahu U. Rangarajan P.N. Methanol expression regulator 1 (Mxr1p) is essential for the utilization of amino acids as the sole source of carbon by the methylotrophic yeast, Pichia pastoris.J. Biol. Chem. 2016; 291: 20588-20601Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). While studying methionine metabolism, a methionine auxotroph (Ppmet6) was generated by replacing the coding region of MET6 encoding MS with a Zeocin expression cassette in the P. pastoris GS115 strain (Fig. 1, A and B). MS deficiency resulted in methionine auxotrophy, and Ppmet6 was unable to grow in a methionine-deficient medium, as expected (Fig. 1C). However, methionine supplementation did not restore the growth of Ppmet6 (Fig. 1D) under conditions in which it readily restored the growth of S. cerevisiae met6 strain (Scmet6) (Fig. 1, E and F). Of the various components tested, adenine when added to YNBD + methionine medium restored growth of Ppmet6 (Fig. 1, G and H) indicating that MS deficiency results in methionine as well as adenine auxotrophy in P. pastoris. To further characterize P. pastoris MS (PpMS),we first examined its subcellular localization. PpMS was expressed as a histidine-tagged protein in E. coli, purified, and injected into rabbits to generate anti-PpMS antibodies that reacted with a protein of ∼86 kDa in the cell lysates of GS115 but not Ppmet6 (Fig. 2, A and B). Immunofluorescence studies revealed that PpMS is present in the nucleus of P. pastoris cells cultured in YPD, YPG, or YPM media (Fig. 2C). To further validate these results, P. pastoris strain expressing chromosomally tagged PpMS-GFP (Pp-PpMSGFP) was constructed, and the localization of GFP fusion protein was visualized by direct examination of GFP fluorescence as well as immunofluorescence of DAPI-stained cells using anti-GFP and anti-PpMS antibodies. The results indicate that PpMSGFP localizes to the nucleus (Fig. 2, D and E). To further confirm nuclear localization of PpMS, P. pastoris GS115 strain was transformed with pGAP-PpMSMyc plasmid expressing c-Myc epitope-tagged PpMS (PpMSMyc) from the GAPDH promoter, and immunofluorescence studies were carried out with anti-c-Myc antibodies. The results indicate that PpMSMyc localizes to the nucleus (Fig. 2F). PpMSMyc expression was confirmed by SDS-PAGE followed by Western blotting of whole-cell extracts using anti-c-Myc as well as anti-PpMS antibodies (Fig. 2G). Because nuclear localization of MS has not been reported in any species thus far and to study the generality of this phenomenon, MS localization was examined in C. albicans, another respiratory yeast, which also exhibits methionine as well as adenine auxotrophy when MET6 is deleted (3.Suliman H.S. Appling D.R. Robertus J.D. The gene for cobalamin-independent methionine synthase is essential in Candida albicans: a potential antifungal target.Arch. Biochem. Biophys. 2007; 467: 218-226Crossref PubMed Scopus (35) Google Scholar). We confirmed that CaMS is immunoreactive to anti-PpMS antibodies by Western blotting (Fig. 2H). Immunofluorescence studies indicate that CaMS localizes to the nucleus in C. albicans as well (Fig. 2I). Because the S. cerevisiae met6 strain does not exhibit adenine auxotrophy, we examined MS localization in this yeast species. ScMS was expressed as a GFP fusion protein (ScMSGFP) in S. cerevisiae met6 strain to generate Sc-ScMSGFP strain. Direct visualization of GFP fluorescence (Fig. 3A) as well as immunofluorescence using anti-GFP antibodies (Fig. 3B) indicates that ScMSGFP is localized to the cytoplasm. Interestingly, when ScMSGFP was expressed in the Ppmet6 strain (Pp-ScMSGFP), it localized to the nucleus as evident from the direct visualization of GFP fluorescence (Fig. 3C) and by immunofluorescence using anti-GFP antibodies (Fig. 3D). When PpMSGFP was expressed in S. cerevisiae (Sc-PpMSGFP), it localized to the cytoplasm as evident from the direct visualization of GFP fluorescence (Fig. 3E) and by immunofluorescence using anti-GFP antibodies (Fig. 3F). Differential subcellular localization of MS in P. pastoris and S. cerevisiae was further confirmed by confocal microscopy (Fig. 3G). Western blot analysis revealed that PpMS is present in the cytoplasm as well as nuclear fractions of P. pastoris, although it is present only in the cytoplasm of S. cerevisiae (Fig. 3H). Taken together, these results indicate that ScMS is cytosolic in S. cerevisiae but nuclear in P. pastoris, and PpMS is nuclear in P. pastoris but cytoplasmic in S. cerevisiae. To understand the mechanism of nuclear localization of PpMS, we examined whether catalytic activity is required for its nuclear localization. In CaMS, the Asp-614 residue in the active site is essential for binding to homocysteine, and consequently, CaMSD612A mutant enzyme possesses only 2% of the native enzyme activity (22.Prasannan P. Suliman H.S. Robertus J.D. Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans.Biochem. Biophys. Res. Commun. 2009; 382: 730-734Crossref PubMed Scopus (11) Google Scholar). Because this aspartate residue is conserved in PpMS (Fig. 4A), we introduced the D612A mutation into PpMSGFP. The Ppmet6 strain expressing PpMSD612AGFP was catalytically inactive as evident from its inability to grow in YNBD medium (Fig. 4B). Direct fluorescence as well as immunofluorescence using anti-GFP antibodies revealed that PpMSD612AGFP localizes to the nucleus (Fig. 4, C–E) indicating that catalytic activity is not required for nuclear localization of PpMS. Analysis of PpMS amino acid sequence using PSort II, PredictNLS, and NetNTS databases indicated that PpMS does not possess a canonical nuclear localization signal (NLS). To examine whether PpMS possesses a non-canonical NLS, we generated pGAPZA vectors encoding PpMSΔ99GFP in which 99 N-terminal amino acids of PpMS are deleted. PpMSΔ99GFP was localized to the nucleus (Fig. 4, C–E), indicating that the 99 N-terminal amino acids are not required for nuclear localization. To examine the role of C-terminal amino acids, we generated pGAPZA vector encoding the PpMSΔ661–768GFP mutant carrying a deletion of 107 C-terminal amino acids, and when expressed in the P. pastoris GS115 strain, it remained in the cytosol (Fig. 4, C–E). Thus, the C-terminal region is essential for nuclear localization of PpMS. Methionine is converted to S-adenosylmethionine by methionine adenosyltransferase (MAT) also known as S-adenosylmethionine synthetase. In mammalian cells, MATI/III was reported to be present in the nucleus, and basic amino acid residues in the C-terminal region of MATI/III are important for nuclear localization (23.Reytor E. Pérez-Miguelsanz J. Alvarez L. Pérez-Sala D. Pajares M.A. Conformational signals in the C-terminal domain of methionine adenosyltransferase I/III determine its nucleocytoplasmic distribution.FASEB J. 2009; 23: 3347-3360Crossref PubMed Scopus (70) Google Scholar). The C-terminal region of PpMS also contains basic amino acid residues, many of which are conserved in ScMS as well as CaMS (Fig. 4F). To examine their role in nuclear localization, we substituted specific arginine/lysine residues present in the C-terminal region of PpMS by alanine. P. pastoris strains expressing PpMSK740AGFP, PpMSR742AGFP, PpMSK759AGFP, or PpMSR762AGFP were generated, and their expression was confirmed by Western blotting with anti-GFP antibodies (Fig. 4G). Analysis of MS-GFP localization by fluorescence microscopy indicates that all the mutants localize to the nucleus except PpMSR742AGFP, which remained in the cytosol (Fig. 4H). Expression of PpMSR742AGFP in Ppmet6 did not result in the restoration of the growth of cells cultured in methionine- and adenine-deficient medium (Fig. 5, A and B). To understand the effect of R742A mutation on enzyme function, the same mutation was introduced into ScMS to generate ScMSR742AGFP. ScMSR742AGFP as well as PpMSR742AGFP were expressed in the Scmet6 strain, and their expression was confirmed by Western blotting using anti-GFP antibodies (Fig. 5C). ScMSR742AGFP and PpMSR742AGFP failed to reverse the methionine auxotrophy of Scmet6 (Fig. 5, D and E) indicating that the R742A mutant is catalytically inactive. To examine the effect of extranuclear localization on enzyme stability and function, we generated Ppmet6 strains expressing PpMSGFP-NES and PpMSGFP-MAS in which a nuclear export signal (NES) or a membrane anchor signal was fused to the C terminus of PpMS, respectively (Fig. 6A). We also generated PpMSGFP-NLS, in which a nuclear localization signal was fused to the C terminus of PpMSGFP (Fig. 6A). As expected, PpMSGFP-NES and PpMSGFP-NLS were targeted to the cytoplasm and nucleus, respectively (Fig. 6B). Although PpMSGFP-MAS was targeted to the plasma membrane, nuclear localization was not completely abrogated (Fig. 6, B and C). Although expression of PpMSGFP-NLS and PpMSGFP-MAS was observed in almost all cells, expression of PpMSGFP-NES was restricted to a small subset of cells (Fig. 6B). Western blot analysis indicated that expression of PpMSGFP-NES was the lowest among the three proteins (Fig. 6D), and as a result, PpMSGFP-NES expression does not result in the reversal of methionine and adenine auxotrophy of Ppmet6. Pp-PpMSGFP-NES strain exhibited severe growth retardation even in medium containing methionine and adenine (Fig. 6, E and F). To understand the mechanism by which the R742A mutation affects enzyme function, structural modeling exercise was carried out to examine the role and significance of Arg-742 at the C-terminal end on the enzyme activity. BLAST search revealed that PpMS shares the highest similarity with CaMS (PDB codes 3PPC, 3PPF, and 3PPH) with a query coverage of 99%, e-value of ∼0, and amino acid sequence identity of 78% (Fig. 7A). 3PPC was used as the base template, and the short stretches of 8 and 6 residues that were missing in the N and C termini, respectively, were modeled based on corresponding regions in 3PPH (Fig. 7A). Superposition of all the structural templates revealed that the substrate-binding site for homocysteine, zinc ion, and the methyl donor have distinct pockets and are seen in between the N- and C-terminal barrel domains (6.Pejchal R. Ludwig M.L. Cobalamin-independent methionine synthase (MetE): a face-to-face double barrel that evolved by gene duplication.PLoS Biol. 2005; 3: e31Crossref PubMed Scopus (96) Google Scholar, 7.Ferrer J.L. Ravanel S. Robert M. Dumas R. Crystal structures of cobalamin-independent methionine synthase complexed with zinc, homocysteine, and methyltetrahydrofolate.J. Biol. Chem. 2004; 279: 44235-44238Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar8.Ubhi D. Kavanagh K.L. Monzingo A.F. Robertus J.D. Structure of Candida albicans methionine synthase determined by employing surface residue mutagenesis.Arch. Biochem. Biophys. 2011; 513: 19-26Crossref PubMed Scopus (6) Google Scholar). In our structural model, Arg-742 is located at a distance of 8 Å from the nearest atom in the substrate-binding pocket and clearly far away from the substrate-binding pocket. Hence, it cannot have any direct effect on the binding of any of the substrates, nor can it be critically involved in stabilizing the pocket. Therefore, we focused our attention on the quaternary structure of cobalamin-independent MS of C. albicans (3PPC) and T. maritima (PDB code 1XPG). The biological assemblies obtained from the PDB database for these two proteins consisted of two subunits of the same polypeptide chain that form a "dimer-like" assembly. With this information, a corresponding dimer-like association was modeled for the PpMS as well, by superposing the whole-length polypeptide chain of our protein with the A and B chains of the structural templates as shown in Fig. 7B. A subsequent energy minimization of the interface of the dimer-like association was also carried out. It was observed that Arg-742 of one subunit makes extensive interactions, mainly hydrogen bonding and ionic in nature, with the residues of the other subunit. Of the many interactions, ionic interaction of Arg-742 with Asp-113 was identified, whose side chains were positioned appropriately for forming a salt bridge (Fig. 6, C and D, and supplemental Fig. 1). Apart from the Arg-742–Asp-113 salt bridge, hydrogen-bonding interactions of Thr-106–Ser-527, Gln-449–Lys-85, Ser-711–Lys-681, Arg-103–Glu-23, Glu-745–Lys-179, Glu-745–Arg-82, and Arg-748–Asp-180 could also be deciphered from the model. The full-length model of the protein in the PDB format is given as supplemental material. The extensive nature of interactions between the two subunits was suggestive of a biological association between the two subunits rather than as an artifact of crystallization. Thus, structural modeling indicated that Arg-742 could play an important role in the stabilization of a dimer-like assembly through an ionic interaction with Asp-113 of a neighboring subunit. To validate these in silico studies, detailed biochemical investigations were carried out. Because R742A mutation results in an inactive enzyme, we examined the effect of the D113R/D113A mutation on enzyme function. PpMSD113AGFP and PpMSD113RGFP were expressed in Scmet6 as well as Ppmet6 strains. Western blot analysis of multiple clones of each mutant indicated that PpMSD113AGFP and PpMSD113RGFP are expressed at lower levels than PpMSGFP (Fig. 8, A–C) suggesting that Asp-113 is essential for the stability of the protein. D113A mutation abrogates enzyme function as evident from the inability of PpMSD113AGFP/PpMSD113RGFP to restore the growth of Scmet6 and Ppmet6 in methionine-deficient media (Fig. 8, D and E