The DNA encoding Rhodobacter sphaeroides phosphoribulokinase (PRK) has been modified to allow ligation into pET-3d. Using the resulting expression plasmid, PRK was overexpressed in Escherichia coli and isolated in milligram quantities. Homogeneous preparations of the enzyme exhibit properties comparable to those of PRK expressed using a previously described pUC19-derived construct [Sandbaken et al., Biochemistry 31, 3715-3719]. Mutagenesis experiments have been designed to produce conservative substitutions that eliminate the carboxyl groups of each of four conserved acidic residues (D42, E131, D169, and E178). Using the newly developed expression system, the resulting PRK variants have been expressed, isolated, and characterized. Expression levels and recoveries upon affinity chromatography purification are similar to the results obtained with wild-type PRK. Apparent substrate affinities of these mutant proteins do not differ greatly from values observed for wild-type PRK. In contrast, these PRK variants display a wide range of Vmax values, ranging from wild-type activity (approximately 200 units/mg; E178A) to levels that are diminished by 4 (D169A) to 5 (D42A, D42N) orders of magnitude. That the large diminutions in catalytic activity are significant and do not merely reflect gross perturbations in protein structure is suggested not only by the modest effects on substrate affinity but also by the allosteric properties of D169A, D42A, and D42N. The activities of these proteins, like that of wild-type PRK, are markedly stimulated by the positive effector NADH. The magnitude of the Vmax perturbations suggests that D42 and D169 are candidates for the role of active site base or activator cation ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Rhodobacter sphaeroides phosphoribulokinase (PRK) binds ATP substrate, as well as spectroscopically active ATP analogs (trinitrophenyl-ATP and ATP gamma S-acetamidoproxyl), to form stable binary complexes. Stoichiometric binding of these nucleotide triphosphates in PRK's substrate site is observed not only with wild-type enzyme but also with D42A and D169A mutants. The demonstration that these mutants contain a full complement of functional substrate binding sites indicates their substantial structural integrity and underscores the significance of their markedly diminished catalytic activity [Charlier et al. (1994) Biochemistry 33, 9343-9350]. Similarly, PRK forms a stable binary complex with the allosteric activator NADH. The negative allosteric effector AMP displaces activator NADH but not substrate from their respective binary complexes with enzyme. When trinitrophenyl-ATP, a fluorescent nucleotide triphosphate that functions as an alternative PRK substrate, forms a binary complex with enzyme, its fluorescence emission is enhanced and lambda max shifted from approximately 557 to 545 nm. Upon formation of a binary PRK-NADH complex, the fluorescence emission of the dinucleotide effector is also enhanced and the lambda max shifted from approximately 460 to 440 nm. PRK forms stable ternary complexes containing NADH and either ATP or trinitrophenyl-ATP. Due to energy transfer, NADH fluorescence in the ternary complex with trinitrophenyl-ATP is markedly quenched, allowing an estimation of the spatial separation between this novel donor/acceptor pair.

The essential photosynthetic enzyme phosphoribulokinase (PRK) is responsible for the conversion of ribulose 5-phosphate (Ru5P) to ribulose 1,5-bisphosphate, the substrate for the CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). We have determined the structure of the octameric bacterial form of PRK to a resolution of 2.5 A. The protein is folded into a seven-member mixed beta-sheet surrounded by alpha-helices, giving the overall appearance of the nucleotide monophosphate family of kinases. Homology with the nucleotide monophosphate kinases suggests a number of amino acid residues that are likely to be important in catalysis and suggests the roles of some amino acid residues that have been mutated prior to the determination of the structure. Further, sequence identity across eukaryotic and prokaryotic species and a calculation of the buried surface area suggests the identity within the octamer of a dimer conserved throughout evolution. The width of the groove leading to the active site is consistent with an oriented molecule of thioredoxin controlling the oxidation state of two cysteines that regulate activity in the eukaryotic enzymes. Although neither Asp 42 nor Asp 169 can be definitively assigned as the catalytic base, the crystal structure suggests the location of a ribulose 5-phosphate binding site and suggests a role for several of the conserved basic residues.

Acetogenic bacteria fix CO or CO2 by a pathway of autotrophic growth called the acetyl-CoA (or Wood) pathway. Key enzymes in the pathway are a methyltransferase, a corrinoid/Fe-S protein, a disulfide reductase, and a carbon monoxide dehydrogenase. This manuscript describes the isolation of the genes that code for the methyltransferase, the two subunits of the corrinoid/Fe-S protein, and the two subunits of carbon monoxide dehydrogenase. These five genes were found to be clustered within an 10-kilobase segment on the Clostridium thermoaceticum genome. The proteins were expressed at up to 5-10% of Escherichia coli cell protein, and isopropyl .8-D-thiogalactopyranoside had no effect on the levels of expression, implying that the C. thermoaceticum inserts contained transcriptional and translational signals that were recognized by E. coli. The methyltransferase is expressed in E. coli in a fully active dimeric form with a specific activity and heat stability similar to the enzyme expressed in C. thermoaceticum. However, both the corrinoid/Fe-S protein and carbon dioxide dehydrogenase, although expressed in high amounts and with identical subunit molecular weights in E. coli, are inactive and less heat stable than are the native enzymes from C. thermoaceticum. sulfides, and 1-3 Zn atoms per dimer (7). The roles of CODH in the pathway are to bind CO (9, 10), a methyl group (11), and CoA (12, 13) and to catalyze the actual synthesis of acetyl-CoA (12).In this manuscript, we report the cloning of the genes for CODH, MeTr, and C/Fe-SP and the expression of the proteins at high levels in Escherichia coli in the absence of any inducer. We have established that these genes are clustered within a 10-kilobase (kb) DNA segment in the C. thermoaceticum genome and that the CODH genes are directly upstream of the 55-kDa subunit of the C/Fe-SP. We suggest that these clustered genes contain promoter-like sequences and translational signals that are recognized by E. coli. MeTr is expressed in E. coli as a heat-stable dimer and is fully active. Both the C/Fe-SP and CODH, although expressed in high amounts by E. coli, are inactive and are much less heat stable than are the active enzymes from C. thermoaceticum.A preliminary report describing the cloning of the C/Fe-SP and MeTr-encoding genes has been published (14). MATERIALS AND METHODSBacterial Strains, Plasmids, and Growth Conditions. C. thermoaceticum, DSM 521, was cultured in 20-liter carboys at 55°C under CO2 as described by Ljungdahl and Andreesen (15). E. coli K-12 strain JM109 (F' traD36 proAB laclqZAM-15/supE44 thi) was cultured on SOB medium (16). Colonies transformed with pUC9 (17) were grown at pH 7.5 at 37°C on LB-ampicillin medium (16) containing 0.8% tryptone, 0.5% yeast extract, 0.5% sodium chloride, and ampicillin at 0.1 mg/ml. 5-Bromo-4-chloro-3-indolyl (3-D-galactopyranoside and isopropyl B-D-thiogalactopyranoside (IPTG) (both from Boehringer Mannheim) were added to the LB-ampicillin medium to final concentrations of 0.032% and 1.6 mM, respectively, for colo...

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.