Richard L. Felts

Structure of Francisella tularensis AcpA PROTOTYPE OF A UNIQUE SUPERFAMILY OF ACID PHOSPHATASES AND PHOSPHOLIPASES C

AcpA is a respiratory burst-inhibiting acid phosphatase from the Centers for Disease Control and Prevention Category A bioterrorism agent Francisella tularensis and prototype of a superfamily of acid phosphatases and phospholipases C. We report the 1.75-Å resolution crystal structure of AcpA complexed with the inhibitor orthovanadate, which is the first structure of any F. tularensis protein and the first for any member of this superfamily. The core domain is a twisted 8-stranded β-sheet flanked by three α-helices on either side, with the active site located above the carboxyl-terminal edge of the β-sheet. This architecture is unique among acid phosphatases and resembles that of alkaline phosphatase. Unexpectedly, the active site features a serine nucleophile and metal ion with octahedral coordination. Structure-based sequence analysis of the AcpA superfamily predicts that the hydroxyl nucleophile and metal center are also present in AcpA-like phospholipases C. These results imply a phospholipase C catalytic mechanism that is radically different from that of zinc metallophospholipases.

Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens

ABSTRACT Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was ∼31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3′ and 5′ nucleoside monophosphates at pH 6. Calculated Kms ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 μmol of Pi/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

Cloning, purification and crystallization of Bacillus anthracis class C acid phosphatase

Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 angstroms. The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flash-annealing. The first data set had a mosaicity of 1.6 degrees and a high-resolution limit of 1.8 angstroms. After flash-annealing, the apparent mosaicity decreased to 0.9 degrees and the high-resolution limit of usable data increased to 1.6 angstroms. This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques.

Crystallization of recombinant Haemophilus influenzae e (P4) acid phosphatase

Haemophilus influenzae infects the upper respiratory tract of humans and can cause infections of the middle ear, sinuses and bronchi. The virulence of the pathogen is thought to involve a group of surface-localized macromolecular components that mediate interactions at the host-pathogen interface. One of these components is lipoprotein e (P4), which is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 angstroms resolution native X-ray diffraction data set. The space group is P4(2)2(1)2, with unit-cell parameters a = 65.6, c = 101.4 angstroms, one protein molecule per asymmetric unit and 37% solvent content. This is the first report of the crystallization of a class C acid phosphatase.

Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis.

Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4(1)2(1)2, with unit-cell parameters a = 61.96, c = 210.78 A. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 A resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

Expression, purification and crystallization of class C acid phosphatases from Francisella tularensis and Pasteurella multocida

Class C nonspecific acid phosphatases are bacterial enzymes that are secreted across the cytoplasmic membrane and hydrolyze a variety of phosphomonoesters at acidic pH. These enzymes are of interest for the development of improved vaccines and clinical diagnostic methods. In one case, the category A pathogen Francisella tularensis, the class C phosphatase plays a role in bacterial fitness. Here, the cloning, expression, purification and crystallization methods for the class C acid phosphatases from F. tularensis and Pasteurella multocida are reported. Crystals of the F. tularensis enzyme diffracted to 2.0 A resolution and belonged to space group C222(1), with one enzyme molecule in the asymmetric unit. Crystals of the P. multocida enzyme diffracted to 1.85 A resolution and belonged to space group C2, with three molecules in the asymmetric unit. Diffraction patterns from crystals of the P. multocida enzyme exhibited multiple interpenetrating reciprocal-space lattices, indicating epitaxial twinning. Despite this aberrance, autoindexing was robust and the data could be satisfactorily processed to 1.85 A resolution using MOSFLM and SCALA.