Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii
© Hwang et al.; licensee BioMed Central Ltd. 2014
Received: 29 May 2014
Accepted: 29 September 2014
Published: 28 October 2014
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.
Thiamine pyrophosphate (TPP) is a cofactor found in virtually all living systems and is important to the function of enzymes in glycolysis, the citric acid cycle (TCA), amino acid biosynthesis, and other metabolic pathways . Many prokaryotes and some eukaryotes (plants and fungi) can synthesize TPP , whereas mammals and other vertebrates require this cofactor in their diet in the form of vitamin B1 (thiamine) . This “thio-“or “sulfur-containing” vitamin consists of a thiazole ring linked to a pyrimidine, with the moieties synthesized separately and then coupled to form thiamine .
Thiazole biosynthesis in bacteria is well characterized and involves a complex chain of oxidative condensation reactions that use 1-deoxy-D-xylulose-5-phosphate, glycine (or tyrosine) and a sulfur source . Sulfur is thought to be derived from cysteine and incorporated into the thiazole ring by a series of enzyme-mediated sulfur transfer steps that include ThiS, ThiF, ThiG, ThiI, and IscS (NifS). In this process, ThiS is adenylated at its C-terminus by ThiF in a mechanism analogous to the activation of ubiquitin. The adenylated ThiS is converted to a thiocarboxylate at the C-terminal glycine residue by sulfur transfer from ThiI and cysteine desulfurase (e.g., IscS, NifS). ThiS thiocarboxylate serves as a sulfur donor for formation of the thiazole ring via thiazole synthase (ThiG).
Molecular details of thiazole ring formation in fungi, yeast and plants are revealed by study of THI4 family proteins. Early work demonstrated that THI4 gene expression is repressed by thiamine and that disruption of this gene in yeast generates strains that are auxotrophic for thiamine, but able to grow in the presence of a thiazole precursor of thiamine . Thus, yeast THI4p was examined for thiazole ring formation and found to catalyze a single turnover reaction in which THI4p Cys205 serves as the source of sulfur to form a thiazole precursor of thiamine with NAD and glycine as co-substrates .
Thiazole biosynthesis in archaea is poorly understood. Most archaea have homologs that cluster to the THI4 family (IPR002922) of proteins, but lack the conserved cysteine residue of the yeast THI4p that is required for sulfur transfer in formation of the thiazole ring and instead have a histidine residue which is well conserved. There are conflicting reports on the function of these archaeal THI4 homologs. Initially, the proteins from Methanocaldococcus jannaschii (MJ0601) and Methanosarcina acetivorans (MA_2851) were reported to convert ribose-1,5-bisphosphate (R15P) into ribulose-1,5-bisphosphate, the substrate of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) . With respect to the experimental details, however, the authors did not measure the assumed activity of a R15P isomerase (which might also be called ribulose-1,5-bisphosphate synthase) but measured stimulation of incorporation of labeled CO2 into an acid-stable compound which was identified as PGA (3-phosphoglyceric acid), the product of RuBisCO. As an additional complication, the starting product 5-phosphoryl-1-pyrophosphate had to be subjected to heat treatment prior to performing the assay. Despite this ambiguity, the conclusions of the initial report with respect to the existence of an archaeal R15P isomerase activity have been confirmed in a subsequent study . These authors directly measured R15P isomerase activity and even put this activity into the wider context of an AMP metabolic pathway (together with RuBisCO and the deoA gene product). However, they assigned the R15P isomerase activity to a completely different protein. This protein, referred to as TK-e2b2, is TK0185 (as retrieved from NCBI using BAD84374, the code provided for this protein) . TK0185 clusters with the e2b2 family (IPR005250) and has no resemblance whatsoever with members of the THI4 family. TK0185 was active in an in vitro R15P isomerase assay. The authors also tested TK0434, the Thermococcus THI4 family member and ortholog of MJ0601, in their in vitro R15P isomerase assay, but did not detect any activity. In addition, they point out that the three enzymes of their AMP metabolic pathway (RuBisCO, e2b2 (TK0185) and DeoA) share a common phylogenetic profile while TK0434 does not. This implies that the original function assignment to MJ0601 and MA_2851 as R15P isomerase may not be correct. Whatever the function of MJ0601 and the other THI4 members with a histidine rather than cysteine may be, there is not the slightest hint that it may be related to thiamine biosynthesis.
Here we report that archaeal THI4 homologs with a conserved catalytic cysteine residue are linked to thiamine biosynthesis. Deletion of the HvThi4 gene homolog (HVO_0665) of Haloferax volcanii was found to confer an auxotrophic requirement for thiamine. The conserved active site cysteine residue of HvThi4 (Cys165) was needed to restore the growth of this thiamine autotroph to wild-type levels. Overall, our results provide new insight that archaeal THI4 family (IPR002922) members with conserved active site cysteine residues are important in thiamine biosynthesis and that most halophilic archaea and ammonia oxidizing archaea (AOA) are likely to use a THI4-type mechanism for sulfur relay to the thiazole ring.
Biochemicals were from Sigma-Aldrich (St. Louis, MO). Other organic and inorganic analytical grade chemicals were from Fisher Scientific (Atlanta, GA). Restriction endonucleases, T4 DNA ligase and Phusion polymerase were from New England Biolabs (Ipswich, MA). Taq DNA polymerase was from Bioline (Taunton, MA). Pfu DNA polymerase was from Stratagene (La Jolla, CA). Desalted oligonucleotides were from Integrated DNA Technologies (Coralville, IA). Agarose for routine analysis of DNA was from Bio-Rad Laboratories (Hercules, CA).
Strains and media
Strains and plasmids used in this study a
Strain, plasmid or primer
Source or reference
F− recA1 endA1 hsdR17(rK − mK +) supE44 thi-1 gyrA relA1
F− ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 rpsL136 dam13::Tn9 xylA5 mtl-1 thi-1 mcrB1 hsdR2
New England Biolabs
endA1 gyrA96(nal R) thi-1 recA1 relA1 lac glnV44 F'[::Tn10 proAB + lacI q Δ(lacZ)M15] hsdR17(rK − mK +)
wild-type isolate DS2 cured of plasmid pHV2
Apr; pBluescript II carries P fdx -pyrE2 with MCS
Apr; Nvr; Hfx. volcanii-E. coli shuttle plasmid, empty vector control
Apr; Nvr; pJAM202c carries P2 rrnA -hvo_1862-strepII
Apr; pTA131-based plasmid for pre-deletion of hvo_0665
Apr; pTA131-based plasmid for Δhvo_0665
Apr; Nvr; pJAM809-derived carries P2 rrnA -hvo_0665-strepII (which replaced hvo_1862-strepII)
Apr; Nvr; pJAM2821-derived carries P2 rrnA -hvo_0665 C165A-strepII
Primer pairs used in this study a
P1: HVO_0665 preKO BamHI RV1
Primers anneal ~500 bp upstream and downstream of hvo_0665; used with H26 genomic DNA as template to generate pJAM2819 (pre-deletion) by ligation into HindIII to BamHI sites of pTA131
5’- ATGAAGCTT AACGCGAGTCTCCTGTGGGCGCTCGG-3'
P2: HVO_0665 HindIII preKO FW1
P4: HVO_0665 3’-inverse
Used to generate plasmid pJAM2820 (Δhvo_0665) by inverse PCR using pJAM2819 as a template
P3: HVO_0665 5’-inverse
P7: HVO_0665 700 bp confirm FW
Anneal ~700 bp upstream and downstream of hvo_0665; used to confirm Δhvo_0665
P8: HVO_0665 700 bp confirm RV
P5: HVO_0665 NdeI
Used to screen for Δhvo_0665 and generate pJAM2821 (in trans complement of Δhvo_0665)
P6: HVO_0665 KpnI
P7: C165A THI4 FW
Used to generate pJAM2822 for synthesis of the site-directed variant HvThi4 C165A
5’- CGCGAACTCACGGCG GTCGACCCCATC-3’
P8: C165A THI4 RV
5’- GATGGGGTCGACCGC CGTGAGTTCGCG-3’
Generation of an Hfx. volcanii Thi4 deletion
For generation of an Hfx. volcanii Δthi4 (Δhvo_0665) strain, a pyrE2-based pop-in/pop-out deletion strategy was used . In brief, plasmid pJAM2819 was constructed by ligation of a 921-bp DNA fragment that was specific for thi4 and its 5’ and 3’ flanking regions (500 bp each) into the BamHI to HindIII sites of the pyrE2-based plasmid pTA131. The resulting pre-deletion plasmid (pJAM2819) was subsequently used as a template to construct the deletion plasmid pJAM2820, which has the thi4 gene deleted by inverse PCR. Plasmid pJAM2820 was used for pyrE2-based recombination and deletion of thi4 on the chromosome of Hfx. volcanii H26. Strain fidelity was confirmed by PCR with primer pairs that annealed within the thi4 ORF (P5/P6) and 700 bp upstream and downstream of thi4 (P7/P8) (Table 2).
Generation of Hfx. volcanii THI4 homolog expression plasmids
The thi4 gene was isolated from Hfx. volcanii genomic DNA by PCR with primers P5/P6 (Table 2). The PCR product was ligated into the NdeI to KpnI sites of plasmid pJAM809, which carries coding sequence for a C-terminal StrepII tag that includes a GT linker (−GTWSHPQPEK, −StrepII) and a ribosomal RNA P2 promoter to drive transcription. The resulting plasmid pJAM2811 was used for trans-expression of HvThi4-StrepII in Hfx. volcanii. For site-directed mutagenesis, plasmid pJAM2811 was isolated from E. coli TOP10 and used as template for PCR with primer pair P7/P8 as indicated in Table 2. Pfu DNA polymerase was used to generate amplicons with the desired single-point mutations as described in the QuikChange Lightning mutagenesis protocol (Stratagene, La Jolla, CA). PCR products were incubated with DpnI at 37°C for 2 h, purified using QIAquick purification kit (Qiagen), and transformed into E. coli XL1-Blue. Plasmid pJAM2812 encoding HvThi4 C165A-StrepII was verified by DNA sequencing.
UniProtKB/Swiss-Prot accession numbers associated with protein sequences analyzed in this study are listed in Additional file 1: Tables S1 and S2. Representative archaeal protein sequences that cluster to known enzymes of thiamine biosynthesis and salvage pathways were retrieved from InterPro  and from GenBank by Basic Local Alignment Search Tool using BlastP (protein-protein BLAST) . Protein sequences were aligned using ClustalW . Prior to dendrogram construction, extensive gaps and N- and C-terminal extensions were removed using BioEdit . A neighborhood-joining tree that best fit the distance data was constructed by pairwise comparisons using MEGA 5 . The mean genetic distance was calculated using p-distance, and gaps were analyzed by pairwise deletion.
Fold recognition and 3D-structural modeling
Phyre2 (Protein Homology/AnalogY Recognition Engine V 2.0) web-based server , was used for fold recognition and model building of HvThi4 (HVO_0665) as well as its homologs, TK0434 and MA_2851. In brief, the primary amino acid sequences were submitted to the Phyre2 server using intensive mode. The Phyre2 threading server combined HHsearch for remote homology detection based on pairwise comparison of hidden Markov models (HMM) with ab initio and multiple-template modeling. The library of known protein structures for comparison was from the Protein Data Bank (PDB) and Structural Classification of Proteins (SCOP) databases. Chimera 1.7  was used as an interface for interactive visualization and analysis of molecular 3D protein structures. Conserved active site residues were identified based on biochemical and structural analysis of ScTHI4p (PDB: 3FPZ) and Neurospora crassa (PDB: 3JSK, also named CyPBP37).
SDS-PAGE and Western blotting analysis
Hfx. volcanii strains were grown in complex medium to stationary-phase (OD600 of 3.0-3.5 in 4 ml ATCC 974 in 13 × 100 mm tubes), harvested by centrifugation (14,500 × g, 2 min at room temperature). Cell pellets were boiled in reducing SDS buffer (2% w/v SDS, 10% v/v glycerol, 5% v/v β-mercaptoethanol, 0.002% w/v bromphenol blue and 62.5 mM Tris–HCl, pH 6.8). Proteins were separated by 12% SDS-PAGE and transferred to Hybond-P polyvinylidene fluoride (PVDF) membranes (GE Healthcare Bio-Sciences, Piscataway, NJ) at 4°C for 2.5 h at 90 V by tank blot. Ponceau Red S Stain was used to confirm equal transfer (Boston Bioproducts, Ashland, MA). StrepII-tagged proteins were detected using unconjungated rabbit anti-StrepII polyclonal antibody (Genscript USA, Piscataway, NJ) and alkaline phosphatase (AP)-linked goat anti-rabbit IgG (H + L) antibody (SouthernBiotech, Birmingham, AL), as primary and secondary antibodies respectively. AP signals were detected by chemiluminescence with CDP-Star according to supplier’s protocol (Applied Biosystems, Carlsbad, CA) and visualized with X-ray film (Hyperfilm, GE Healthcare Bio-Sciences). Equivalent protein loading was further confirmed by staining samples similarly separated by SDS-PAGE in gel with Coomassie Blue R-250.
Results and discussion
Two distinct mechanisms for incorporating sulfur into the thiazole ring of thiamine predicted in select archaea by genomic reconstruction
An updated genomic comparison of thiamine metabolism homologs of select archaea was performed (Additional file 1). Based on this analysis, Hfx. volcanii and related archaea were predicted to use bacterial related enzymes for synthesis of the pyrimidine moiety (HMP-PP, 4-amino-hydroxymethyl-2-methylpyrimidine pyrophosphate) of thiamine. By contrast, the mechanism used for formation of 4-methyl-5-(β-hydroxyethyl) thiazole phosphate (HET-P or THZ-P) containing the thiazole ring was less clear for these organisms. A critical step in formation of the thiazole ring is the incorporation of sulfur. Genomic reconstruction detected two distinct mechanisms for sulfur transfer to synthesize the thiazole ring including: a bacterial ThiF-ThiS related system (UbaA-SAMP) and a yeast THI4p-type mechanism (HvThi4, HVO_0665). However, a conundrum existed in that the UbaA-SAMP system lacked a predicted interaction partner (such as the bacterial ThiG) and that archaeal THI4 homologs (such as MJ0601 and MA_2851) are reported to function as R15P isomerases , thus being unrelated to thiamine biosynthesis. To shed some light into this enigma, we analyzed the members of the Thi4 protein family in more detail.
Haloarchaea and other select archaea have THI4 homologs with a conserved active site cysteine
The ScTHI4p Cys205 residue is highly conserved among eukaryotic THI4 homologs, is required for THI4 activity, and is thought to be used as a sulfur donor in an iron-mediated sulfur transfer reaction to form the thiazole ring, based on MS-based detection of a dehydroalanine residue at this position after a single-turnover reaction . Since the majority of archaeal THI4 homologs lack the conserved active site cysteine, these enzymes are not predicted to use a yeast THI4p-type mechanism of thiazole biosynthesis. However, a subset of archaeal THI4 homologs was detected that displayed not only an overall amino acid sequence similarity to ScTHI4, but also harbored a cysteine residue analogous to the ScTHI4 active site Cys205. For example, the Hfx. volcanii THI4 homolog (HvThi4, HVO_0665) had a conserved active site cysteine residue (Cys165) and 31% overall amino acid sequence identity to ScTHI4p with a BLAST query coverage of 92% and E value of 3e-26. The subset of archaeal THI4 homologs with the conserved active site cysteine residue was found to cluster (Figure 1B) and included THI4 homologs of all of the haloarchaea and ammonium-oxidizing Thaumarchaeota examined as well as the crenarchaeote Aeropyrum pernix, and select euryarchaeota including Pyrococcus sp. strain NA2 and P. yayanosii strain CH1 as well as Methanobacterium sp. strains AL-21 and SWAN-1 (Figure 1B). These select species of Pyrococcus and Methanobacterium had two THI4 homologs (one with a conserved active site cysteine residue, clustering with the haloarchaeal homologs, and the other with a conserved histidine residue, clustering with the other His-containing family members (Figure 1B).
Generation of a thiamine auxotroph of Haloferax volcanii by deletion of the THI4 homolog
Based on these results, we suggest that Thi4 functions as a suicide thiamine thiazole synthase in Hfx. volcanii similarly to yeast . If so, build-up of inactivated HvThi4 in its dehydroalanine form is anticipated. When expressed in Hfx. volcanii, the primary isoform of HvThi4 and its C165A variant that accumulated in cells was observed at 40 kDa, which is in relatively close agreement to the theoretical molecular mass of 33.6 kDa for an acidic protein (pI 4.43) likely to bind less SDS than the protein standards used to estimate its molecular mass . However, at least four protein bands of 60 to 150 kDa were detected for the wild type form of HvThi4 that were not observed for the C165A variant. If HvThi4 uses a suicide mechanism, the “dehydroalanine” form of HvThi4 Cys165 generated after sulfur transfer to the thiazole ring precursor would be inactivate and, thus, the protein ‘reagent’ may be susceptible to aggregation or some type of post-translational modification that could facilitate its elimination from the cell. The sampylation system may fulfill this role in archaea by covalently attaching ubiquitin-like SAMP moieties to Thi4 and targeting it for proteasome-mediated degradation . Alternatively, Hfx. volcanii cells may have a mechanism to minimize overproduction of thiamine through modification of Thi4.
Availability of supporting data
All supporting data are included as an additional Supplementary Materials file deposited in LabArchives, LLC under the dataset link DOI:10.6070/H4959FJX.
Thanks to S. Shanker and the staff at UF ICBR for Sanger DNA sequencing. The authors acknowledge funding awarded to JM-F through the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (Grant DE-FG02-05ER15650) for work on ubiquitin-fold protein function in biosynthetic pathways and the National Institutes of Health (Grant R01 GM57498) for work on E1-like mechanisms.
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