MF7000913 Bi-functional enzyme 6j2l X-ray 2.17 Homodimer Shigella flexneri 31235255 Wang Y, Zhang F, Nie Y, Shang G, Zhang H Biochem. Biophys. Res. Commun. 2019 2 516 540-545 Histidine biosynthesis, which is absent in animals, was shown to be highly conserved among gram-negative bacteria, thus making it an attractive target for antibiotic design. There are many fusion forms of enzymes in the histidine biosynthetic pathway and people still have limited knowledge about their domain organizations and catalytic mechanisms, due to the lack of structural information. Here we report the first crystal structure of Shigella flexneri bi-functional enzyme HisIE (SfHisIE) that functions in the 2nd and 3rd steps in the histidine biosynthetic pathway. This structure shows that HisIE exists as dimers with two loops (fusion loop) connecting the individual dimer of HisE and HisI in its N-terminus and C-terminus respectively. Our mutagenesis study shows mutations in this fusion loop are lethal for bacteria indicating the advantage of gene fusion in Histidine biosynthesis. Structural analysis revealed several highly conserved residues in the putative ligand binding grooves of HisE and HisI, showing an evolutionarily conserved catalytic mechanism shared among gram negative-bacteria. GO:0005524 ATP binding GO:0004635 phosphoribosyl-AMP cyclohydrolase activity GO:0004636 phosphoribosyl-ATP diphosphatase activity GO:0005737 cytoplasm 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 bifunctional protein HisIE Shigella flexneri P37793 2 201 98% MLTEQQRRELDWEKTDGLMPVIVQHAVSGEVLMLGYMNPEALDKTIESGKVTFFSRTKQRLWIKGETSGNFLNVVSIAPDCDNDTLLVLANPIGPTCHKGTSSCFGNTAHQWLFLYQLEQLLAERKYADPETSYTAKLYASGTKRIAQKVGEEGVETALAATVHDRFELTNEASDLMYHLLVLLQDQDLDLTTVIENLHKRHQ 203 secondary structure helix 2 9 secondary structure helix 37 48 secondary structure helix 111 124 secondary structure helix 132 141 secondary structure helix 141 162 secondary structure helix 164 186 secondary structure helix 189 200 secondary structure strand 18 24 secondary structure strand 30 36 secondary structure strand 51 54 secondary structure strand 59 62 secondary structure strand 70 78 secondary structure strand 85 92 pfam PF01503 Phosphoribosyl-ATP pyrophosphohydrolase 115 202 B Histidine biosynthesis bifunctional protein HisIE Shigella flexneri P37793 3 201 98% MLTEQQRRELDWEKTDGLMPVIVQHAVSGEVLMLGYMNPEALDKTIESGKVTFFSRTKQRLWIKGETSGNFLNVVSIAPDCDNDTLLVLANPIGPTCHKGTSSCFGNTAHQWLFLYQLEQLLAERKYADPETSYTAKLYASGTKRIAQKVGEEGVETALAATVHDRFELTNEASDLMYHLLVLLQDQDLDLTTVIENLHKRHQ 203 secondary structure helix 2 8 secondary structure helix 37 48 secondary structure helix 111 124 secondary structure helix 125 127 secondary structure helix 137 140 secondary structure helix 141 163 secondary structure helix 164 186 secondary structure helix 189 200 secondary structure strand 18 24 secondary structure strand 30 36 secondary structure strand 51 53 secondary structure strand 60 62 secondary structure strand 70 78 secondary structure strand 85 92 pfam PF01503 Phosphoribosyl-ATP pyrophosphohydrolase 115 202 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