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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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Related in: MedlinePlus

The pathway forde novobiosynthesis of thiamine in the archaeonHaloferax volcaniiis an apparent hybrid of bacterial and yeast pathways. Yeast and bacterial like steps are shaded by purple and blue, respectively, with associated enzymes of thiamine biosynthetic indicated in text with similar color coding. Hfx. volcanii homologs are indicated by gene locus tag in green. Question mark (?) designated enzyme is yet unassigned. THI4-SH specifies the catalytic cysteine side chain. THI4-C = CH indicates the dehydroalanine form of the enzyme after sulfur transfer. The sulfur atom associated with formation of the thiazole ring is highlighted in red. In the Hfx. volcanii model for thiamine biosynthesis, the thiazole moiety of thiamine (4-methyl-5-(β-hydroxyethyl)thiazole phosphate; HET-P or THZ-P) is generated by a yeast-like mechanism. HvThi4 (HVO_0665) converts nicotinamide adenine dinucleotide (NAD) and glycine to ADP-thiazole (ADT), which predicted to be hydrolyzed to THZ-P by a yet to be identified NUDIX-type hydrolase [29]. The remaining steps of thiamine biosynthesis are related to bacterial systems. In particular, the PurM-like AIR synthetase HVO_1557 is predicted to form 5-amino-1-(5-phospho-D-ribosyl)imidazole (synonym 5-aminoimidazole ribotide; AIR), which serves as a substrate for a ThiC-like S-adenosyl-methionine (SAM)-dependent HMP-P synthase (HVO_2154) in the generation of 4-amino-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P). Once generated, HMP-P is phosphorylated by a bacterial ThiD-type HMP-P kinase (HVO_2666) which is also conserved in the N-terminal domains (Ntd) of yeast THI20 and THI21. Thiamine-phosphate synthase homologs of the ThiE-type (HVO_2668) and ThiN-type (HVO_0662) are predicted to condense THZ-P with HMP-PP to form thiamine monophosphate (TMP). TMP is then phosphorylated to TPP by a proposed bacterial ThiL-type thiamine-monophosphate kinase (HVO_1861).
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Fig6: The pathway forde novobiosynthesis of thiamine in the archaeonHaloferax volcaniiis an apparent hybrid of bacterial and yeast pathways. Yeast and bacterial like steps are shaded by purple and blue, respectively, with associated enzymes of thiamine biosynthetic indicated in text with similar color coding. Hfx. volcanii homologs are indicated by gene locus tag in green. Question mark (?) designated enzyme is yet unassigned. THI4-SH specifies the catalytic cysteine side chain. THI4-C = CH indicates the dehydroalanine form of the enzyme after sulfur transfer. The sulfur atom associated with formation of the thiazole ring is highlighted in red. In the Hfx. volcanii model for thiamine biosynthesis, the thiazole moiety of thiamine (4-methyl-5-(β-hydroxyethyl)thiazole phosphate; HET-P or THZ-P) is generated by a yeast-like mechanism. HvThi4 (HVO_0665) converts nicotinamide adenine dinucleotide (NAD) and glycine to ADP-thiazole (ADT), which predicted to be hydrolyzed to THZ-P by a yet to be identified NUDIX-type hydrolase [29]. The remaining steps of thiamine biosynthesis are related to bacterial systems. In particular, the PurM-like AIR synthetase HVO_1557 is predicted to form 5-amino-1-(5-phospho-D-ribosyl)imidazole (synonym 5-aminoimidazole ribotide; AIR), which serves as a substrate for a ThiC-like S-adenosyl-methionine (SAM)-dependent HMP-P synthase (HVO_2154) in the generation of 4-amino-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P). Once generated, HMP-P is phosphorylated by a bacterial ThiD-type HMP-P kinase (HVO_2666) which is also conserved in the N-terminal domains (Ntd) of yeast THI20 and THI21. Thiamine-phosphate synthase homologs of the ThiE-type (HVO_2668) and ThiN-type (HVO_0662) are predicted to condense THZ-P with HMP-PP to form thiamine monophosphate (TMP). TMP is then phosphorylated to TPP by a proposed bacterial ThiL-type thiamine-monophosphate kinase (HVO_1861).

Mentions: Here we resolve the enigma of how sulfur is incorporated into the thiazole ring of thiamine in archaea such as Hfx. volcanii and propose a model in which bacterial related enzymes are used for synthesis of the pyrimidine moiety (HMP-PP) and eukaryotic enzymes are used for formation of the thiazole derivative (HET-P or THZ-P) of thiamine (Figure 6). While genomic reconstruction suggested Hfx. volcanii used two distinct mechanisms for sulfur incorporation into the thiazole ring, in vivo analysis revealed that only the yeast Thi4-type (and not the bacterial ThiF-ThiS) system was required. In particular, the conserved catalytic cysteine residue (Cys165) of the Hfx. volcanii THI4p homolog (HvThi4) was found to be essential in the trans complementation of a Δthi4 mutant for growth in the absence of thiamine or THZ. Cluster analysis of THI4p homologs revealed a clear separation of archaeal homologs with a conserved active site cysteine residue, such as HvThi4, from those having a histidine residue in its place (the latter of which are implicated in functions distinct from thiamine biosynthesis [6,7,22]). Based on this distinction, we propose a yeast-type THI4p mechanism, which uses an active site cysteine residue for sulfur incorporation into the thiazole ring of thiamine, is relatively widespread in archaea including halophilic archaea of the phylum Euryarchaeota and ammonium oxidizing archaea of the phylum Thaumarchaeota.Figure 6


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)

The pathway forde novobiosynthesis of thiamine in the archaeonHaloferax volcaniiis an apparent hybrid of bacterial and yeast pathways. Yeast and bacterial like steps are shaded by purple and blue, respectively, with associated enzymes of thiamine biosynthetic indicated in text with similar color coding. Hfx. volcanii homologs are indicated by gene locus tag in green. Question mark (?) designated enzyme is yet unassigned. THI4-SH specifies the catalytic cysteine side chain. THI4-C = CH indicates the dehydroalanine form of the enzyme after sulfur transfer. The sulfur atom associated with formation of the thiazole ring is highlighted in red. In the Hfx. volcanii model for thiamine biosynthesis, the thiazole moiety of thiamine (4-methyl-5-(β-hydroxyethyl)thiazole phosphate; HET-P or THZ-P) is generated by a yeast-like mechanism. HvThi4 (HVO_0665) converts nicotinamide adenine dinucleotide (NAD) and glycine to ADP-thiazole (ADT), which predicted to be hydrolyzed to THZ-P by a yet to be identified NUDIX-type hydrolase [29]. The remaining steps of thiamine biosynthesis are related to bacterial systems. In particular, the PurM-like AIR synthetase HVO_1557 is predicted to form 5-amino-1-(5-phospho-D-ribosyl)imidazole (synonym 5-aminoimidazole ribotide; AIR), which serves as a substrate for a ThiC-like S-adenosyl-methionine (SAM)-dependent HMP-P synthase (HVO_2154) in the generation of 4-amino-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P). Once generated, HMP-P is phosphorylated by a bacterial ThiD-type HMP-P kinase (HVO_2666) which is also conserved in the N-terminal domains (Ntd) of yeast THI20 and THI21. Thiamine-phosphate synthase homologs of the ThiE-type (HVO_2668) and ThiN-type (HVO_0662) are predicted to condense THZ-P with HMP-PP to form thiamine monophosphate (TMP). TMP is then phosphorylated to TPP by a proposed bacterial ThiL-type thiamine-monophosphate kinase (HVO_1861).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: The pathway forde novobiosynthesis of thiamine in the archaeonHaloferax volcaniiis an apparent hybrid of bacterial and yeast pathways. Yeast and bacterial like steps are shaded by purple and blue, respectively, with associated enzymes of thiamine biosynthetic indicated in text with similar color coding. Hfx. volcanii homologs are indicated by gene locus tag in green. Question mark (?) designated enzyme is yet unassigned. THI4-SH specifies the catalytic cysteine side chain. THI4-C = CH indicates the dehydroalanine form of the enzyme after sulfur transfer. The sulfur atom associated with formation of the thiazole ring is highlighted in red. In the Hfx. volcanii model for thiamine biosynthesis, the thiazole moiety of thiamine (4-methyl-5-(β-hydroxyethyl)thiazole phosphate; HET-P or THZ-P) is generated by a yeast-like mechanism. HvThi4 (HVO_0665) converts nicotinamide adenine dinucleotide (NAD) and glycine to ADP-thiazole (ADT), which predicted to be hydrolyzed to THZ-P by a yet to be identified NUDIX-type hydrolase [29]. The remaining steps of thiamine biosynthesis are related to bacterial systems. In particular, the PurM-like AIR synthetase HVO_1557 is predicted to form 5-amino-1-(5-phospho-D-ribosyl)imidazole (synonym 5-aminoimidazole ribotide; AIR), which serves as a substrate for a ThiC-like S-adenosyl-methionine (SAM)-dependent HMP-P synthase (HVO_2154) in the generation of 4-amino-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P). Once generated, HMP-P is phosphorylated by a bacterial ThiD-type HMP-P kinase (HVO_2666) which is also conserved in the N-terminal domains (Ntd) of yeast THI20 and THI21. Thiamine-phosphate synthase homologs of the ThiE-type (HVO_2668) and ThiN-type (HVO_0662) are predicted to condense THZ-P with HMP-PP to form thiamine monophosphate (TMP). TMP is then phosphorylated to TPP by a proposed bacterial ThiL-type thiamine-monophosphate kinase (HVO_1861).
Mentions: Here we resolve the enigma of how sulfur is incorporated into the thiazole ring of thiamine in archaea such as Hfx. volcanii and propose a model in which bacterial related enzymes are used for synthesis of the pyrimidine moiety (HMP-PP) and eukaryotic enzymes are used for formation of the thiazole derivative (HET-P or THZ-P) of thiamine (Figure 6). While genomic reconstruction suggested Hfx. volcanii used two distinct mechanisms for sulfur incorporation into the thiazole ring, in vivo analysis revealed that only the yeast Thi4-type (and not the bacterial ThiF-ThiS) system was required. In particular, the conserved catalytic cysteine residue (Cys165) of the Hfx. volcanii THI4p homolog (HvThi4) was found to be essential in the trans complementation of a Δthi4 mutant for growth in the absence of thiamine or THZ. Cluster analysis of THI4p homologs revealed a clear separation of archaeal homologs with a conserved active site cysteine residue, such as HvThi4, from those having a histidine residue in its place (the latter of which are implicated in functions distinct from thiamine biosynthesis [6,7,22]). Based on this distinction, we propose a yeast-type THI4p mechanism, which uses an active site cysteine residue for sulfur incorporation into the thiazole ring of thiamine, is relatively widespread in archaea including halophilic archaea of the phylum Euryarchaeota and ammonium oxidizing archaea of the phylum Thaumarchaeota.Figure 6

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