MF7000914 MtHISN2, a bifunctional enzyme 7bgm X-ray 1.60 Homodimer Medicago truncatula 33958623 Witek W, Sliwiak J, Ruszkowski M Sci Rep 2021 1 11 9647 The second and third steps of the histidine biosynthetic pathway (HBP) in plants are catalyzed by a bifunctional enzyme-HISN2. The enzyme consists of two distinct domains, active respectively as a phosphoribosyl-AMP cyclohydrolase (PRA-CH) and phosphoribosyl-ATP pyrophosphatase (PRA-PH). The domains are analogous to single-domain enzymes encoded by bacterial hisI and hisE genes, respectively. The calculated sequence similarity networks between HISN2 analogs from prokaryotes and eukaryotes suggest that the plant enzymes are closest relatives of those in the class of Deltaproteobacteria. In this work, we obtained crystal structures of HISN2 enzyme from Medicago truncatula (MtHISN2) and described its architecture and interactions with AMP. The AMP molecule bound to the PRA-PH domain shows positioning of the N1-phosphoribosyl relevant to catalysis. AMP bound to the PRA-CH domain mimics a part of the substrate, giving insights into the reaction mechanism. The latter interaction also arises as a possible second-tier regulatory mechanism of the HBP flux, as indicated by inhibition assays and isothermal titration calorimetry. GO:0005524 ATP binding GO:0046872 metal ion binding GO:0004635 phosphoribosyl-AMP cyclohydrolase activity GO:0004636 phosphoribosyl-ATP diphosphatase activity GO:0008652 amino acid biosynthetic process GO:0000105 histidine biosynthetic process 2 1 Homooligomeric enzymes Homodimeric enzymes All chains according to the most probable oligomerization state stored in PDBe were considered. A Histidine biosynthesis hisIE protein Medicago truncatula A0A072U2X9 49 264 76% MALSNVHMLQSARGFQKCNLSFSSHGYPRRGYRTNHLAFASMHTSDPKVDSLLDSVKWDNKGLAVAIAQNVDTGAILMQGFANREAVATTISSRKATFYSRSRSSLWTKGETSNNFINVHDVFLDCDRDSIIYLGKPDGPTCHTGAETCYYTPVFDLLKEEEVEGNKLALTSLYALESTISQRKAEVVEENGKPSWTKRLLLNDKLLCSKIREEANELCETLENNEDKSRTASEMADVLYHAMVLLALKDVKVEEVLQVLRQRFSKSGIEEKRSRPTQKSVEN 283 secondary structure helix 2 10 secondary structure helix 38 49 secondary structure helix 110 114 secondary structure helix 126 141 secondary structure helix 150 157 secondary structure helix 158 179 secondary structure helix 182 205 secondary structure helix 207 219 secondary structure strand 19 25 secondary structure strand 31 37 secondary structure strand 52 55 secondary structure strand 60 63 secondary structure strand 72 80 secondary structure strand 85 92 secondary structure strand 105 108 pfam PF01503 Phosphoribosyl-ATP pyrophosphohydrolase 174 263 B Histidine biosynthesis hisIE protein Medicago truncatula A0A072U2X9 49 265 76% MALSNVHMLQSARGFQKCNLSFSSHGYPRRGYRTNHLAFASMHTSDPKVDSLLDSVKWDNKGLAVAIAQNVDTGAILMQGFANREAVATTISSRKATFYSRSRSSLWTKGETSNNFINVHDVFLDCDRDSIIYLGKPDGPTCHTGAETCYYTPVFDLLKEEEVEGNKLALTSLYALESTISQRKAEVVEENGKPSWTKRLLLNDKLLCSKIREEANELCETLENNEDKSRTASEMADVLYHAMVLLALKDVKVEEVLQVLRQRFSKSGIEEKRSRPTQKSVEN 283 secondary structure helix 2 9 secondary structure helix 38 49 secondary structure helix 109 114 secondary structure helix 126 140 secondary structure helix 150 157 secondary structure helix 158 179 secondary structure helix 182 204 secondary structure helix 207 219 secondary structure strand 19 25 secondary structure strand 31 37 secondary structure strand 52 55 secondary structure strand 60 63 secondary structure strand 72 80 secondary structure strand 85 92 secondary structure strand 105 108 pfam PF01503 Phosphoribosyl-ATP pyrophosphohydrolase 174 263 Indirect evidence The full structure participates in mutual synergistic folding. Histidine biosynthesis bifunctional protein HisIE and MtHISN2 are proteins with with discrete and directly interacting pyrophosphohydrolase and cyclohydrolase domains. They form a tight dimer with large buried interface. The dimer is formed by two mutually swapped polypeptide chains, forming a bilobial protein. The existence of a monomeric form of either PRA-PH or PRA-CH domain is highly improbable. The dimeric form is consistent with the size-exclusion elution profile. In case of HisIE dimerization is very important for catalytic activity, since its catalytic pocket is formed by both protomers. Arg201 from one monomer is required to stabilize helix 4 in the other monomer, thus making it suitable for substrate binding (PMID:31235255, PMID:33958623). A N/A B N/A MF7000912 MF7000913 MF7000914