title: ThiN as a Versatile Domain of Transcriptional Repressors and Catalytic Enzymes of Thiamine Biosynthesis.
abstract: Thiamine biosynthesis is commonly regulated by a riboswitch mechanism; however, the enzymatic steps and regulation of this pathway in archaea are poorly understood.Haloferax volcanii, one of the representative archaea, uses a eukaryote-like Thi4 (thiamine thiazole synthase) for the production of the thiazole ring and condenses this ring with a pyrimidine moiety synthesized by an apparent bacterium-like ThiC (2-methyl-4-amino-5-hydroxymethylpyrimidine [HMP] phosphate synthase) branch. Here we found that archaeal Thi4 and ThiC were encoded by leaderless transcripts, ruling out a riboswitch mechanism. Instead, a novel ThiR transcription factor that harbored an N-terminal helix-turn-helix (HTH) DNA binding domain and C-terminal ThiN (TMP synthase) domain was identified. In the presence of thiamine, ThiR was found to repress the expression ofthi4andthiCby a DNA operator sequence that was conserved across archaeal phyla. Despite having a ThiN domain, ThiR was found to be catalytically inactive in compensating for the loss of ThiE (TMP synthase) function. In contrast, bifunctional ThiDN, in which the ThiN domain is fused to an N-terminal ThiD (HMP/HMP phosphate [HMP-P] kinase) domain, was found to be interchangeable for ThiE function and, thus, active in thiamine biosynthesis. A conserved Met residue of an extended α-helix near the active-site His of the ThiN domain was found to be important for ThiDN catalytic activity, whereas the corresponding Met residue was absent and the α-helix was shorter in ThiR homologs. Thus, we provide new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveal that thiamine biosynthesis in archaea is regulated by a transcriptional repressor, ThiR, and not by a riboswitch.IMPORTANCEThiamine pyrophosphate (TPP) is a cofactor needed for the enzymatic activity of many cellular processes, including central metabolism. In archaea, thiamine biosynthesis is an apparent chimera of eukaryote- and bacterium-type pathways that is not well defined at the level of enzymatic steps or regulatory mechanisms. Here we find that ThiN is a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea. Our study provides new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveals that archaeal thiamine biosynthesis is regulated by a ThiN domain transcriptional repressor, ThiR, and not by a riboswitch.
authors.affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA.
authors.forename: Sungmin
authors.initials: S
authors.lastname: Hwang
authors.affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Cordova
authors.affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA.
authors.forename: Merna
authors.initials: M
authors.lastname: Abdo
authors.affiliation: Computational Biology Group, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Pfeiffer
authors.affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA jmaupin@ufl.edu.|Genetics Institute, University of Florida, Gainesville, Florida, USA.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Maupin-Furlow
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D019641:DNA, Archaeal / Q000235:genetics; D019848:Gene Expression Regulation, Archaeal / Q000502:physiology*; D019622:Haloferax volcanii / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D011687:Purines / Q000737:chemistry / Q000378:metabolism; D013831:Thiamine / Q000096:biosynthesis* / Q000737:chemistry; D014157:Transcription Factors / Q000502:physiology*
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D011687:Purines; D014157:Transcription Factors; D013831:Thiamine
keywords: archaea; cofactor metabolism; thiamine biosynthesis; transcription factor (ThiR)