Structural Snapshots of Escherichia coli Histidinol Phosphate Phosphatase along the Reaction Pathway

Abstract

HisB from Escherichia coli is a bifunctional enzyme catalyzing the sixth and eighth steps of L-histidine biosynthesis. The N-terminal domain (HisB-N) possesses histidinol phosphate phosphatase activity, and its crystal structure shows a single domain with fold similarity to the haloacid dehalogenase (HAD) enzyme family. HisB-N forms dimers in the crystal and in solution. The structure shows the presence of a structural Zn 2؉ ion stabilizing the conformation of an extended loop. Two metal binding sites were also identified in the active site. Their presence was further confirmed by isothermal titration calorimetry. HisB-N is active in the presence of Mg 2؉ , Mn 2؉ , Co 2؉ , or Zn 2؉ , but Ca 2؉ has an inhibitory effect. We have determined structures of several intermediate states corresponding to snapshots along the reaction pathway, including that of the phosphoaspartate intermediate. A catalytic mechanism, different from that described for other HAD enzymes, is proposed requiring the presence of the second metal ion not found in the active sites of previously characterized HAD enzymes, to complete the second half-reaction. The proposed mechanism is reminiscent of two-Mg 2؉ ion catalysis utilized by DNA and RNA polymerases and many nucleases. The structure also provides an explanation for the inhibitory effect of Ca 2؉ .The histidine biosynthetic pathway serves as a model system for better understanding of the fundamental metabolic, physiological, and genetic processes in bacteria (1). This pathway is identical in both Escherichia coli and Salmonella typhimurium and has been thoroughly characterized (1, 2). The sixth and eighth steps of histidine biosynthesis are catalyzed by imidazole glycerol phosphate dehydratase (IGPD, 3 EC 4.2.1.19) and histidinol phosphate phosphatase (HPase, EC.3.1.3.15) respectively (1) (Scheme 1). In protobacteria, including E. coli and S. typhimurium, the IGPD and HPase activities are encoded by a single gene (3-5), whereas in archaea, eukarya, and most bacteria they are encoded by two separate genes (3). The bifunctional HisB enzyme has been proposed to be the result of a fusion of two independent cistrons that occurred recently in evolution (3).Biochemical and genetic studies of the HisB enzyme together suggest that both of its enzymatic activities are independent of one another and reside in separate domains (6 -8). The HPase activity is found within the N-terminal domain (residues 1-167, HisB-N), whereas the C-terminal domain (residues 168 -356) exhibits the IGPD activity. The phosphatase activity requires the presence of metal ions such as Mg 2ϩ , Mn 2ϩ , Co 2ϩ , or Zn 2ϩ but is inhibited by calcium (9). Based on the presence of four invariant aspartic acid residues, HisB has been classified as a member of the haloacid dehalogenase-like hydrolase (HAD) family within the "DDDD" superfamily of aspartyl-phosphate utilizing phosphohydrolases/phosphotransferases (10, 11). The phosphoryl transfer catalyzed by phosphotransferases and phosphatases from the HAD family occurs via a phosphoa...

Panoramic view of a superfamily of phosphatases through substrate profiling

“…To examine the propensity of HADSF members for hydrolyzing the substrates in the library, the frequency of each substrate being considered a "hit" was computed. Markedly, pNPP was among the most common substrates (ranked eighth) and was used by 30.4% of the enzymes tested. This is instructive, given the fact that pNPP is commonly used to test for generic phosphatase activity.…”

Structural Studies of Medicago truncatula Histidinol Phosphate Phosphatase from Inositol Monophosphatase Superfamily Reveal Details of Penultimate Step of Histidine Biosynthesis in Plants

“…The differences in reaction mechanism are reflected by the very diverse turnover rates of IMPase-like and DDDD HPPs. The k cat value for MtHPP is 3.6 s Ϫ1 , whereas HisB-N converts HOLP to HOL nearly 3 orders of magnitude faster, with k cat ϭ 2.14 ϫ 10 3 s Ϫ1 (83). HisB-N also has a ϳ5 times lower K m (54 M) than does MtHPP, making the latter, and all other examples of IMPaselike HPPs, relatively inefficient enzymes.…”

Structural and functional analysis of the phosphoryl transfer reaction mediated by the human small C‐terminal domain phosphatase, Scp1

“…32 Additionally, phosphoryl-aspartate intermediates have been detected by radioactivity measurements in DNA 3 0 phosphatase Tpp1, 33 and in a recent study, Ca 2þ ions were shown to have an inhibitory effect and induce the accumulation of a phosphoryl-aspartate intermediate detected by mass spectrometry in histidinol phosphate phosphatase. 34 …”