Thiamine biosynthesis is commonly regulated by a riboswitch mechanism; however, the 21 enzymatic steps and regulation of this pathway in archaea are poorly understood. Haloferax 22 volcanii, one of the representative archaea, uses a yeast-like Thi4 (thiamine thiazole synthase) 23 for production of the thiazole ring and condenses this ring with a pyrimidine moiety synthesized 24 by an apparent bacterial-like ThiC (HMP-phosphate synthase) branch. Here we found the 25 archaeal Thi4 and ThiC were encoded by leaderless transcripts, ruling out a riboswitch 26 mechanism. Instead, a novel ThiR transcription factor was identified that harbored an N-terminal 27 HTH-DNA binding domain and C-terminal ThiN (TMP synthase) do... More
Thiamine biosynthesis is commonly regulated by a riboswitch mechanism; however, the 21 enzymatic steps and regulation of this pathway in archaea are poorly understood. Haloferax 22 volcanii, one of the representative archaea, uses a yeast-like Thi4 (thiamine thiazole synthase) 23 for production of the thiazole ring and condenses this ring with a pyrimidine moiety synthesized 24 by an apparent bacterial-like ThiC (HMP-phosphate synthase) branch. Here we found the 25 archaeal Thi4 and ThiC were encoded by leaderless transcripts, ruling out a riboswitch 26 mechanism. Instead, a novel ThiR transcription factor was identified that harbored an N-terminal 27 HTH-DNA binding domain and C-terminal ThiN (TMP synthase) domain. In the presence of 28 thiamine, ThiR was found to repress the expression of thi4 and thiC by a DNA operator 29 sequence that was conserved across archaeal phyla. Despite having a ThiN domain, ThiR was 30 found catalytically inactive in compensating for loss of ThiE (TMP synthase) function. By 31 contrast, a bifunctional ThiDN, in which the ThiN domain is fused to an N-terminal ThiD 32 (HMP/HMP~P kinase) domain, was found interchangeable for ThiE function and, thus, active in 33 thiamine biosynthesis. A conserved Met residue of an extended α-helix near the active site His 34 of the ThiN domain was found important for ThiDN catalytic activity; whereas, the corresponding 35 Met residue was absent and the α-helix was shorter in ThiR homologs. Thus, we provide a new 36 insight into residues that distinguish catalytic from non-catalytic ThiN domains and reveal that 37 thiamine biosynthesis in archaea is regulated by a transcriptional repressor ThiR, not by a 38 riboswitch.
