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Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii.

Hwang S, Cordova B, Chavarria N, Elbanna D, McHugh S, Rojas J, Pfeiffer F, Maupin-Furlow JA - BMC Microbiol. (2014)

Bottom Line: 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).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.Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611-0700, USA. sungminhwang@ufl.edu.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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TheHaloferax volcanii thi4gene and its in-frame deletion. (A) Schematic representation of the thi4 gene on the genome of Hfx. volcanii DS2. HVO_0662 encodes a ThiN homolog with a predicted N-terminal helix-turn-helix (HTH) DNA binding domain. The PCR primer pairs (P1/P2 and P3/P4) used to generate the thi4 gene deletion and the PCR primer pairs (P5/P6 and P7/P8) used to screen for the thi4 gene deletion are indicated. (B) PCR products generated for the Δthi4 (Δhvo_0665) mutant and parent (H26, wt) strains using primer pairs P5/P6 and P7/P8 as indicated. Size reduction with primer pair P7/P8 and the absence of a signal with primer pair P5/P6 confirm the deletion in the Δthi4 strain.
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Fig3: TheHaloferax volcanii thi4gene and its in-frame deletion. (A) Schematic representation of the thi4 gene on the genome of Hfx. volcanii DS2. HVO_0662 encodes a ThiN homolog with a predicted N-terminal helix-turn-helix (HTH) DNA binding domain. The PCR primer pairs (P1/P2 and P3/P4) used to generate the thi4 gene deletion and the PCR primer pairs (P5/P6 and P7/P8) used to screen for the thi4 gene deletion are indicated. (B) PCR products generated for the Δthi4 (Δhvo_0665) mutant and parent (H26, wt) strains using primer pairs P5/P6 and P7/P8 as indicated. Size reduction with primer pair P7/P8 and the absence of a signal with primer pair P5/P6 confirm the deletion in the Δthi4 strain.

Mentions: To further understand the molecular mechanisms used by archaea with predicted ThiS/ThiF- and THI4-type pathways to synthesize the thiazole ring of thiamine, a genetic strategy was used in which Hfx. volcanii strains with targeted deletions of gene homologs in the two predicted pathways were analyzed for growth in the absence of supplied thiamine. Consistent with our metabolic reconstruction results, Hfx. volcanii has the metabolic capacity for thiamine biosynthesis as shown by its ability to grow on minimal medium in the absence of an exogenous source of thiamine [23]. Hfx. volcanii is also demonstrated to synthesize key metabolic enzymes that are TPP-dependent including the 2-oxoacid (α-ketoacid): ferredoxin oxidoreductases used to mediate the oxidative decarboxylation of pyruvate and 2-oxoglutarate [24] and three 2-oxoacid dehydrogenases used under nitrate-respirative conditions [25] including one involved in growth on isoleucine [26]. The ΔubaA mutant required to analyze the ThiS/ThiF-type pathway was already available from previous study [11]. A Δthi4 (HVO_0665) mutant strain (NC1011) was generated for this study (Figure 3) using a pyrE based pop-in/pop-out strategy similar to generation of the ΔubaA mutant (Table 1).Figure 3


Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii.

Hwang S, Cordova B, Chavarria N, Elbanna D, McHugh S, Rojas J, Pfeiffer F, Maupin-Furlow JA - BMC Microbiol. (2014)

TheHaloferax volcanii thi4gene and its in-frame deletion. (A) Schematic representation of the thi4 gene on the genome of Hfx. volcanii DS2. HVO_0662 encodes a ThiN homolog with a predicted N-terminal helix-turn-helix (HTH) DNA binding domain. The PCR primer pairs (P1/P2 and P3/P4) used to generate the thi4 gene deletion and the PCR primer pairs (P5/P6 and P7/P8) used to screen for the thi4 gene deletion are indicated. (B) PCR products generated for the Δthi4 (Δhvo_0665) mutant and parent (H26, wt) strains using primer pairs P5/P6 and P7/P8 as indicated. Size reduction with primer pair P7/P8 and the absence of a signal with primer pair P5/P6 confirm the deletion in the Δthi4 strain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4215014&req=5

Fig3: TheHaloferax volcanii thi4gene and its in-frame deletion. (A) Schematic representation of the thi4 gene on the genome of Hfx. volcanii DS2. HVO_0662 encodes a ThiN homolog with a predicted N-terminal helix-turn-helix (HTH) DNA binding domain. The PCR primer pairs (P1/P2 and P3/P4) used to generate the thi4 gene deletion and the PCR primer pairs (P5/P6 and P7/P8) used to screen for the thi4 gene deletion are indicated. (B) PCR products generated for the Δthi4 (Δhvo_0665) mutant and parent (H26, wt) strains using primer pairs P5/P6 and P7/P8 as indicated. Size reduction with primer pair P7/P8 and the absence of a signal with primer pair P5/P6 confirm the deletion in the Δthi4 strain.
Mentions: To further understand the molecular mechanisms used by archaea with predicted ThiS/ThiF- and THI4-type pathways to synthesize the thiazole ring of thiamine, a genetic strategy was used in which Hfx. volcanii strains with targeted deletions of gene homologs in the two predicted pathways were analyzed for growth in the absence of supplied thiamine. Consistent with our metabolic reconstruction results, Hfx. volcanii has the metabolic capacity for thiamine biosynthesis as shown by its ability to grow on minimal medium in the absence of an exogenous source of thiamine [23]. Hfx. volcanii is also demonstrated to synthesize key metabolic enzymes that are TPP-dependent including the 2-oxoacid (α-ketoacid): ferredoxin oxidoreductases used to mediate the oxidative decarboxylation of pyruvate and 2-oxoglutarate [24] and three 2-oxoacid dehydrogenases used under nitrate-respirative conditions [25] including one involved in growth on isoleucine [26]. The ΔubaA mutant required to analyze the ThiS/ThiF-type pathway was already available from previous study [11]. A Δthi4 (HVO_0665) mutant strain (NC1011) was generated for this study (Figure 3) using a pyrE based pop-in/pop-out strategy similar to generation of the ΔubaA mutant (Table 1).Figure 3

Bottom Line: 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).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.Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611-0700, USA. sungminhwang@ufl.edu.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus