Skip to main content

Structural and mechanistic characterization of L-histidinol phosphate phosphatase from the polymerase and histidinol phosphatase family of proteins

Ghodge SV, Fedorov AA, Fedorov EV, Hillerich B, Seidel R, Almo SC, Raushel FM (2013) Biochemistry 52, 1101-12. PMCID: PMC3570733

Members in the AH Bridging Project, Protein Core, and Structure Core determined that an EFI AH superfamily target functions as a phosphatase during the biosynthesis of histidine.  Through this effort the EFI groups determined which structural features contribute to substrate recognition and catalysis thereby increasing our understanding of what elements form the basis of specificity in multi-functional enzyme superfamilies.  

Abstract

l-Histidinol phosphate phosphatase (HPP) catalyzes the hydrolysis of l-histidinol phosphate to l-histidinol and inorganic phosphate, the penultimate step in the biosynthesis of l-histidine. HPP from the polymerase and histidinol phosphatase (PHP) family of proteins possesses a trinuclear active site and a distorted (β/α)7-barrel protein fold. This group of enzymes is closely related to the amidohydrolase superfamily of enzymes. The mechanism of phosphomonoester bond hydrolysis by the PHP family of HPP enzymes was addressed. Recombinant HPP from Lactococcus lactis subsp. lactis that was expressed in Escherichia coli contained a mixture of iron and zinc in the active site and had a catalytic efficiency of 103 M–1 s–1. Expression of the protein under iron-free conditions resulted in the production of an enzyme with a 2 order of magnitude improvement in catalytic efficiency and a mixture of zinc and manganese in the active site. Solvent isotope and viscosity effects demonstrated that proton transfer steps and product dissociation steps are not rate-limiting. X-ray structures of HPP were determined with sulfate, l-histidinol phosphate, and a complex of l-histidinol and arsenate bound in the active site. These crystal structures and the catalytic properties of variants were used to identify the structural elements required for catalysis and substrate recognition by the HPP family of enzymes within the amidohydrolase superfamily.

Link to PubMed »

Abstract ImageAbstract Image

Scheme 1Scheme 1

Figure 1Figure 1. Cartoon depicting the metal ligation scheme for members of the amidohydrolase superfamily and the PHP family that bind two and three divalent metal ions, respectively.

Figure 2Figure 2. Sequence similarity network of proteins in cog 1387 at an E value of 10–20 created using Cytoscape (http://www.cytoscape.org). Each node (sphere) represents a single sequence, and each edge (line) represents the pairwise connection between two sequences with the most significant BLAST E value (better than 10–20). Lengths of edges are not significant, except for tightly clustered groups, which are more closely related than sequences with only a few connections. The nodes were assigned colors as follows: blue for authentic HPP enzymes colocalized with other genes involved in the biosynthesis of l-histidine; green for gene products that possess all the sequence motifs characteristic of the HPP enzymes but not found colocalized with other l-histidine biosynthetic genes present in these organisms; red for gene products that possess all the sequence motifs characteristic of an authentic HPP, but the organism lacks a majority of the genes required for l-histidine biosynthesis; gray for protein sequences that are significantly similar in sequence to authentic HPP enzymes but lack certain sequence elements critical for HPP activity; and orange for proteins that are not HPP enzymes. Specific HPP enzymes mentioned in this paper are indicated by numbers 1–10. These proteins are identified by their locus tags: L37351 (1), MCCL_0344 (2), BBR47_00270 (3), BCE_1533 (4), BcerKBAB4_1335 (5), BSU29620 (6), BH3206 (7), GK2799 , SMU_1486c (9), TTHA0331 (10), and LMOh7858_0629 (11). The HPP enzymes characterized in this study are shown as large spheres, and enzymes whose crystal structures are available in the Protein Data Bank are indicated as diamonds. Other crystal structures available for enzymes in this cog are indicated as diamonds and include (A) YcdX from E. coli (Protein Data Bank entries 1m65, 1m68, and 1pb0) and (B) the N-terminal PHP domain of DNA polymerase X from Deinococcus radiodurans (Protein Data Bank entry 2w9m).

Figure 3Figure 3. pH–rate profile for the hydrolysis of histidinol phosphate by HPP from L. lactis at 22 ± 1 °C. (A) Variation of log kcat/Km vs pH. The solid line represents a fit of the data to eq 4. (B) Variation of log kcat vs pH. The solid line represents a fit of the data to eq 3.

Figure 4Figure 4. Effect of solvent viscosity on kcat (A) and kcat/Km (B) for the hydrolysis of histidinol phosphate by HPP at pH 8.6.

Figure 5Figure 5. Three-dimensional structure of HPP from L. lactis with sulfate bound at the active site The α-helices are colored red, β-sheets green, loops cyan, and active site Zn2+ ions orange.

Figure 6Figure 6. Active site structure of HPP from L. lactis. The three Zn2+ ions in the active site are shown as orange spheres, and the enzyme residues serving as ligands to the metal ions are colored light blue.

Figure 7Figure 7. Representative electron density map for the active site of HPP complexed with Zn2+ and l-histidinol arsenate and contoured at 1.5σ. The figure was produced with PyMOL.(26) The details of the interactions between l-histidinol arsenate and the active site are described in the text.

Figure 8Figure 8. Product and inhibitory complexes in the active site of HPP. Zinc ions are presented as orange spheres, while the enzyme residues are colored blue. (A) Inorganic phosphate and l-histidinol bound at the active site of HPP from L. lactis. Inorganic phosphate is colored orange and l-histidinol light green. (B) l-Histidinol–arsenate ester, a substrate mimic, bound at the active site of HPP from L. lactis. Arsenate is colored brown and l-histidinol dark green.

Figure 9Figure 9. Overlay of the HPP·Zn·HAR and HPP·Zn·HOL·HPO4 structures. The former is shown in dark shades, while the latter is shown in lighter shades. Metal ions are colored orange and enzyme residues blue. l-Histidinol is colored green and water/hydroxide red; arsenate and phosphate are colored brown and orange, respectively.

Scheme 2Scheme 2

Figure 10Figure 10. Primary sequence alignment of HPPs from various organisms. L37351 is the HPP from L. lactis subsp. lactis Il1403 characterized in this study. The organisms from which the other HPPs were derived were Br. brevis NBRC 100599 (BBR47_00270), M. caseolyticus JCSC5402 (MCCL_0344), B. cereus ATCC10987 (BCE_1533), B. weihenstephanensis KBAB4 (BcerKBAB4_1335), B. subtilis subsp. subtilis str. 168 (BSU29620), B. halodurans C-125 (BH3206), G. kaustophilus HTA426 (GK2799), S. mutans UA159 (SMU_1486c), T. thermophilus HB8 (TTHA0331), and Li. monocytogenes str. 4b h7858 (LMOh7858_0629). The primary sequence of L37351 shown here is the actual sequence of the recombinant protein characterized in this study, while the remaining sequences are from the NCBI database. Metal-binding residues are colored red, while the conserved residues interacting with the substrate are colored green. The β-sheets observed in the available crystal structures of the three HPP enzymes are highlighted in gray.

Reprinted with permission from Biochemistry. Copyright 2013 American Chemical Society.