Crystal structure of a sulfur carrier protein complex found in the cysteine biosynthetic pathway of Mycobacterium tuberculosis.
ABSTRACT: The structure of the protein complex CysM-CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 A resolution. CysM (Rv1336) is a PLP-containing beta-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 A resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM-CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF-ThiS and MoeB-MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.
Project description: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 DeltacsdA and DeltasufS 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.
Project description:A new pathway for cysteine biosynthesis has been elucidated in Mycobacterium tuberculosis. This pathway involves a protein-bound thiocarboxylate (CysO-SH) as the sulfide donor, similar to thiamin biosynthesis. Cysteine synthase M (CysM) catalyzes the addition of cysteine to the carboxy terminus of the protein-bound thiocarboxylate to generate a CysO-cysteine adduct. A protease, Mec+, hydrolyzes the CysO-cysteine adduct to release cysteine and regenerate CysO. Mec+ contains a JAMM motif, and this work provides the first functional characterization of the JAMM motif in prokaryotes. MoeZ, a paralogue of ThiF, has been shown to transfer sulfur onto CysO.
Project description:We have determined the crystal structure of the Escherichia coli ThiS-ThiF protein complex at 2.0 A resolution. ThiS and ThiF are bacterial proteins involved in the synthesis of the thiazole moiety of thiamin. ThiF catalyzes the adenylation of the carboxy terminus of ThiS and the subsequent displacement of AMP catalyzed by ThiI-persulfide to give a ThiS-ThiI acyl disulfide. Disulfide interchange, involving Cys184 on ThiF, then generates the ThiS-ThiF acyl disulfide, which functions as the sulfur donor for thiazole formation. ThiS is a small 7.2 kDa protein that structurally resembles ubiquitin and the molybdopterin biosynthetic protein MoaD. ThiF is a 27 kDa protein with distinct sequence and structural similarity to the ubiquitin activating enzyme E1 and the molybdopterin biosynthetic protein MoeB. The ThiF-ThiS structure clarifies the mechanism of the sulfur transfer chemistry involved in thiazole biosynthesis.
Project description:Here we provide the first detailed biochemical study of a noncanonical E1-like enzyme with broad specificity for cognate ubiquitin-like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding, and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1-like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed that ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1-like complex. Importantly, the thioester was an apparent precursor to Ubl protein modification but not sulfur mobilization. Comparative modeling to MoeB/ThiF guided the discovery of key residues within the adenylation domain of UbaA that were needed to bind ATP as well as residues that were specifically needed to catalyze the downstream reactions of sulfur mobilization and/or Ubl protein modification. UbaA was also found to be Ubl-automodified at lysine residues required for early (ATP binding) and late (sulfur mobilization) stages of enzyme activity revealing multiple layers of autoregulation. Cysteine residues, distinct from the canonical E1 'active site' cysteine, were found important in UbaA function supporting a model that this noncanonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1-like thioester and cysteine persulfide(s) intermediates to form.
Project description:Protein modifiers are involved in diverse biological processes and regulate the activity or function of target proteins by covalently conjugating to them. Although ubiquitin and a number of ubiquitin-like protein modifiers (Ubls) in eukaryotes have been identified, no protein modifier has been found in prokaryotes; thus, their evolutionary origin remains a puzzle. To infer the evolutionary relationships between the protein modifiers and sulfur carrier proteins, we solved the solution NMR structure of the Urm1 (ubiquitin-related modifier-1) protein from Saccharomyces cerevisiae. Both structural comparison and phylogenetic analysis of the ubiquitin superfamily, with emphasis on the Urm1 family, indicate that Urm1 is the unique "molecular fossil" that has the most conserved structural and sequence features of the common ancestor of the entire superfamily. The similarities of 3D structure and hydrophobic and electrostatic surface features between Urm1 and MoaD (molybdopterin synthase small subunit) suggest that they may interact with partners in a similar manner, and similarities between Urm1-Uba4 and MoaD-MoeB establish an evolutionary link between ATP-dependent protein conjugation in eukaryotes and ATP-dependent cofactor sulfuration.
Project description:A covalently linked protein--protein conjugate between ThiF and ThiS thiocarboxylate was found in a partially purified coexpressed ThiF/ThiS protein mixture by using Fourier transform mass spectrometry. The Cys-184 of ThiF and the C terminus of ThiS thiocarboxylate were identified to be involved in the formation of this complex by using both mutagenesis and chemical modification methods. A complementation study of Escherichia coli thiF(-) using thiF(C184S) suggests that this conjugate is an essential intermediate involved in the biosynthesis of the thiazole moiety of thiamin. This ThiF/ThiS conjugate is the first characterized example of a unique acyldisulfide intermediate in a biosynthetic system. This protein conjugate is also an example of an ubiquitin-E1 like protein-protein conjugate in prokaryotes and supports a strong evolutionary link between thiamin biosynthesis and the ubiquitin conjugating system.
Project description:Thiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms.Based on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant.Based on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.
Project description:The gram-positive human pathogen Staphylococcus aureus is often isolated with media containing potassium tellurite, to which it has a higher level of resistance than Escherichia coli. The S. aureus cysM gene was isolated in a screen for genes that would increase the level of tellurite resistance of E. coli DH5alpha. The protein encoded by S. aureus cysM is sequentially and functionally homologous to the O-acetylserine (thiol)-lyase B family of cysteine synthase proteins. An S. aureus cysM knockout mutant grows poorly in cysteine-limiting conditions, and analysis of the thiol content in cell extracts showed that the cysM mutant produced significantly less cysteine than wild-type S. aureus SH1000. S. aureus SH1000 cannot use sulfate, sulfite, or sulfonates as the source of sulfur in cysteine biosynthesis, which is explained by the absence of genes required for the uptake and reduction of these compounds in the S. aureus genome. S. aureus SH1000, however, can utilize thiosulfate, sulfide, or glutathione as the sole source of sulfur. Mutation of cysM caused increased sensitivity of S. aureus to tellurite, hydrogen peroxide, acid, and diamide and also significantly reduced the ability of S. aureus to recover from starvation in amino acid- or phosphate-limiting conditions, indicating a role for cysteine in the S. aureus stress response and survival mechanisms.
Project description:The biosynthesis of thiamin pyrophosphate (TPP) in prokaryotes, as represented by the Escherichia coli and the Bacillus subtilis pathways, is summarized in this review. The thiazole heterocycle is formed by the convergence of three separate pathways. First, the condensation of glyceraldehyde 3-phosphate and pyruvate, catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (Dxs), gives 1-deoxy-D-xylulose 5-phosphate (DXP). Next, the sulfur carrier protein ThiS-COO- is converted to its carboxyterminal thiocarboxylate in reactions catalyzed by ThiF, ThiI, and NifS (ThiF and IscS in B. subtilis). Finally, tyrosine (glycine in B. subtilis) is converted to dehydroglycine by ThiH (ThiO in B. subtilis). Thiazole synthase (ThiG) catalyzes the complex condensation of ThiS-COSH, dehydroglycine, and DXP to give a thiazole tautomer, which is then aromatized to carboxythiazole phosphate by TenI (B. subtilis). Hydroxymethyl pyrimidine phosphate (HMP-P) is formed by a complicated rearrangement reaction of 5-aminoimidazole ribotide (AIR) catalyzed by ThiC. ThiD then generates hydroxymethyl pyrimidine pyrophosphate. The coupling of the two heterocycles and decarboxylation, catalyzed by thiamin phosphate synthase (ThiE), gives thiamin phosphate. A final phosphorylation, catalyzed by ThiL, completes the biosynthesis of TPP, the biologically active form of the cofactor. This review reviews the current status of mechanistic and structural studies on the enzymes involved in this pathway. The availability of multiple orthologs of the thiamin biosynthetic enzymes has also greatly facilitated structural studies, and most of the thiamin biosynthetic and salvage enzymes have now been structurally characterized.
Project description:Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.