Crystal Structure of Cryptosporidium parvum Pyruvate Kinase

Abstract

Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303–320) of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.

An overview of structure, function, and regulation of pyruvate kinases

“…Crystal structures of Cp PyK in the apo‐form ( 4DRS ) and in the holo‐form in complex with ADP, Mg 2+ , and K + have been determined ( 6P0Y ) (Table S5). Notably, in these structures, a unique covalently cross‐linked dimer is observed wherein Cys26 of each monomer forms a disulfide bridge with Cys312 of the other at the A–A interface . Notably, both Cys26 and Cys312 are unique in the Cp PyK sequence, and no other disulfide‐linked PyK dimer has been reported.…”

“…As an intestinal pathogen, C. parvum has adapted to the microaerophilic environment and relies exclusively on glycolysis for ATP production . The C. parvum genome contains a single pyk gene encoding the glycolytic form of the enzyme, which demonstrates distinct functional and structural properties as compared to other PyKs . Crystal structures of Cp PyK in the apo‐form ( 4DRS ) and in the holo‐form in complex with ADP, Mg 2+ , and K + have been determined ( 6P0Y ) (Table S5).…”

“…Notably, the Cp PyK sequence has an unusually high content of 21 Cys residues whereas most PyKs contain only 10–15. Cp PyK enzyme remains active under reducing conditions . Additional structural and functional studies are necessary to characterize this distinct PyK.…”

“…86 The C. parvum genome contains a single pyk gene encoding the glycolytic form of the enzyme, which demonstrates distinct functional and structural properties as compared to other PyKs. 87,88 Crystal structures of CpPyK in the apo-form (4DRS) 88 and in the holo-form in complex with ADP, Mg 2+ , and K + have been determined (6P0Y) (- Table S5). Notably, in these structures, a unique covalently cross-linked dimer is observed wherein Cys26 of each monomer forms a disulfide bridge with Cys312 of the other at the A-A interface.…”

“…Notably, in these structures, a unique covalently cross-linked dimer is observed wherein Cys26 of each monomer forms a disulfide bridge with Cys312 of the other at the A-A interface. 88 Notably, both Cys26 and Cys312 are unique in the CpPyK sequence, and no other disulfide-linked PyK dimer has been reported. These disulfide bonds may contribute to the increased structural stability of CpPyK dimers as suggested from the calculated ΔG diss values for the dimers in apo-and holo-CpPyK crystal structures (Table S6).…”