A Catalytic Mechanism Revealed by the Crystal Structures of the Imidazolonepropionase from Bacillus subtilis

Abstract

Imidazolonepropionase (EC 3.5.2.7) catalyzes the third step in the universal histidine degradation pathway, hydrolyzing the carbon-nitrogen bonds in 4-imidazolone-5-propionic acid to yield N-formimino-L-glutamic acid. Here we report the crystal structures of the Bacillus subtilis imidazolonepropionase and its complex at 2.0-Å resolution with substrate analog imidazole-4-acetic acid sodium (I4AA). The structure of the native enzyme contains two domains, a TIM (triose-phosphate isomerase) barrel domain with two insertions and a small ␤-sandwich domain. The TIM barrel domain is quite similar to the members of the ␣/␤ barrel metallo-dependent hydrolase superfamily, especially to Escherichia coli cytosine deaminase. A metal ion was found in the central cavity of the TIM barrel and was tightly coordinated to residues His-80, His-82, His-249, Asp-324, and a water molecule. X-ray fluorescence scan analysis confirmed that the bound metal ion was a zinc ion. An acetate ion, 6 Å away from the zinc ion, was also found in the potential active site. In the complex structure with I4AA, a substrate analog, I4AA replaced the acetate ion and contacted with Arg-89, Try-102, Tyr-152, His-185, and Glu-252, further defining and confirming the active site. The detailed structural studies allowed us to propose a zinc-activated nucleophilic attack mechanism for the hydrolysis reaction catalyzed by the enzyme.The histidine degradation pathway is highly conserved from prokaryotes to eukaryotes (1, 2). In bacteria, the histidine degradation pathway is operated by the hut (histidine utilization) genes, and the Bacillus subtilis hut operon encodes proteins in the order of HutP, HutH, HutU, HutI, HutG, HutM (3-6).There (Fig. 1A); 4) N-formimino-L-glutamic acid ϩ H 2 O 3 glutamate ϩ HCONH 2 (catalyzed by formiminoglutamase, EC 3.5.3.8).Among the four enzymes, the first three-dimensional structure determined was the 2.1-Å resolution crystal structure of histidase or HutH from Pseudomonas putida by Schulz and co-workers in 1999 (7,8). It was found that there was a polypeptide modification, forming a novel catalytically essential electrophilic group. The second structure was of urocanase or HutU, also from P. putida, and the structure was determined by the same group who solved the histidase in 2004 (9). The third structure solved was the last enzyme in the histidine degradation pathway, formiminoglutamase or HutG from Vibrio cholerae, which was determined by Wu et al. 5 and deposited in the Protein Data Bank under accession number 1XFK. The imidazolonepropionase or HutI is the only enzyme left in this pathway that has no published structural information so far, although some properties of this enzyme have been studied in Salmonella typhimurium (10), Pseudomonas fluorescens ATCC 11299 (11), rat liver (12), and other organisms during 1960s and 1970s. The maximal activity of this enzyme occurred at pH 7.4 with a narrow pH optimum, and its Michaelis constant was calculated to be 7 M (12). It was also mentioned in the literature that 1 mM EDTA could...

Regulation of the Histidine Utilization (Hut) System in Bacteria

“…The enzyme from Bacillus subtilis has 421 amino acids per monomer (170), which is slightly larger than the predicted Pseudomonas enzyme. Its substrate, IP, is unstable, with a half-life of about 20 to 25 min (125,128).…”

“…Early kinetic studies used IP produced in situ from urocanate in the presence of highly purified urocanase. As a result, the characterization of IPase lagged behind that of the other Hut enzymes, and IPase was the last of the enzymes to have its crystal structure determined (161,170). Although the details of the reaction mechanism and the nature of the active site metal remain somewhat unclear, two facts are well established: the reaction is essential for utilization of histidine as a carbon source (74,148), and IPase is a hydrolase that cleaves the ring to yield formiminoglutamate, in a different reaction from the nonenzymatic cleavage that yields formylisoglutamine (128).…”

Structure of dihydropyrimidinase from Sinorhizobium meliloti CECT4114: New features in an amidohydrolase family member

“…1UN7; Vincent et al, 2004), imidazalonepropionase (PDB ID. 2BB0; Yu et al, 2006), and enamidase (PDB ID. 2VUM, Kress et al, 2008).…”

Logical Identification of an Allantoinase Analog (puuE) Recruited from Polysaccharide Deacetylases

“…Glu 36 could stabilize the oxyanion intermediate through H-bond and donate a proton to the N-3 atom of the substrate, creating a good leaving group. In this way Glu 36 would play the same role of the catalytic glutamate of imidazolonepropionase (49), an enzyme unrelated to PuuE acting on a substrate similar to allantoin.…”