Identification of the phosphoribulokinase sugar phosphate binding domain

Abstract

A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (form I; NADH dependent) has been expressed in and purified to homogeneity from Escherichia coli that harbor the prkA gene in the plasmid pKP1565b. Restriction digestion of the phosphoribulokinase-encoding plasmid produces a tractable 450 bp fragment that encodes amino acid residues 28-179, which include a region (residues 42-54) highly conserved among phosphoribulokinase proteins. Using overlap extension polymerase chain reaction methodology, directed mutagenesis was performed to produce mutant proteins in which basic residues in this conserved region were replaced by neutral amino acids. Lysine-53, implicated by affinity labeling studies, has been replaced by methionine; little effect on substrate binding or catalysis is apparent. In contrast, when histidine-45 is replaced by asparagine, a 40-fold increase in the Km for ribulose 5-phosphate results; a 200-fold increase results when arginine-49 is replaced by glutamine. Implication of this region as part of the sugar phosphate binding site is compatible with previous results that indicate targeting by an ATP analogue containing a reactive functionality esterified to the gamma-phosphoryl group. The phosphoribulokinase reaction involves a single in-line phosphoryl transfer, requiring that the gamma-phosphoryl of ATP be closely juxtaposed to the bound cosubstrate. It follows that any reactive group attached to the gamma-phosphoryl in a nucleotide analogue that is bound to PRK in the absence of the cosubstrate will be favorably positioned to modify the sugar phosphate binding site.

The Crystal Structure of Phosphoribulokinase from Rhodobacter sphaeroides Reveals a Fold Similar to That of Adenylate Kinase

“…On the basis of the structural similarity surrounding the P-loop among these proteins and the locations of conserved amino acids known to be important to catalysis, it is assumed that residues in the vicinity of the P-loop and the bound sulfate molecule form the active site of PRK. Site-directed mutagenesis data are consistent with this conclusion (9)(10)(11). The active site is delineated by the main -sheet, the P-loop that projects into the active site, and the three helices B, E, and F ( Figure 3).…”

Functional Evaluation of Invariant Arginines Situated in the Mobile Lid Domain of Phosphoribulokinase

“…While K m effects do not always correlate well with changes in affinity, effects of the type and magnitude observed for R173Q are frequently used to implicate an amino acid in substrate binding (30). The 10 2 -fold effect observed with R173Q is comparable in magnitude to the effect previously reported for R49Q (13). However, it is clear that, if the K m data are interpreted to suggest that R49 and R173 are both involved in binding of Ru5P's C5 phosphoryl group, there would have to be considerable movement of the lid domain to close the active site cavity and bring R49 and R173 into proximity.…”

“…While these acidic amino acids could function as activator cation ligands or as a catalytic base, precise assignments of function to each of these residues have not been made. However, a specific function in sugar phosphate substrate binding has been proposed for prokaryotic PRK's R49 (13) or, correspondingly, eukaryotic PRK's R64 (14), on the basis of the large K m Ru5P effects that are observed upon mutagenesis of this arginine.…”

A RuBisCO-mediated carbon metabolic pathway in methanogenic archaea

“…2f; Supplementary Fig. 7b,d)2526272829. Mutagenesis and structural studies on RsPRK have also identified other residues responsible for Ru5P binding (His45 and Lys165) and ATP binding (Arg168).…”