Overproduction of Anabaena 7120 ribulose-bisphosphate carboxylase/oxygenase in Escherichia coli.

As a prerequisite to protein engineering, we have overexpressed the rbcLS operon of the cyanobacterium Anabaena 7120, in Escherichia coli. The operon encodes the large and small subunits of ribulose-bisphosphate carboxylase/oxygenase (Rubisco). Levels of active enzyme exceed 6% of soluble protein. We noted an apparent third gene, an unidentified open reading frame (URF) referred to here as rbcX, in the 558-bp intergenic space between the large and small subunit encoding genes. The URF, rbcX, has no known function. High-level production of Rubisco activity from the rbc operon in E. coli required simultaneous overproduction of the GroESL chaperonins under a regimen of limited growth, in contrast to more modest conditions which suffice for efficient production of the Anacystis nidulans cyanobacterial Rubisco. Deletion of rbcX or inversion of the rbcL-rbcS order did not enhance expression levels. The recombinant Rubisco, purified to near homogeneity, exhibited functional properties [Km(ribulose-P2), kcat, transition-state-analogue binding stoichiometry/exchange, and specificity factor] essentially identical to those of the enzyme obtained from Anabaena.

Different modes of action of inhibitors of bacterial D-amino acid transaminase. A target enzyme for the design of new antibacterial agents.

D-Amino acid transaminase from Bacillus sphaericus shows a deuterium kinetic isotope effect (VH/VD) between 2 and 3 in the transamination of alpha-protio- or alpha-deuterio-D-alanine and alpha-ketoglutarate, suggesting that alpha-proton abstraction is at least partially rate-limiting for this reaction. This transaminase also catalyzes a beta-elimination reaction with substrates such as beta-fluoroalanine with no detectable deuterium isotope effect (VH/BD = 1). These results, taken together with previous work (Soper, T. S., and Manning, J. M. (1978) Biochemistry 17, 3377-3384) suggest that the rate-limiting step in the beta-elimination reaction is solvolysis of an alpha-aminoacrylate-pyridoxal-P Schiff's base intermediate. D-Cycloserine is an active site titrant of D-amino acid transaminase. Inactivation by cycloserine can be completely reversed by dialysis against pyridoxal phosphate at neutral pH. Gabaculine is also an efficient inhibitor of this enzyme and possesses some antibacterial activity. The latter two inhibitors probably act by sequestration of the coenzyme rather than by alkylation of the protein as with the beta-halo derivatives of D-alanine.

Inactivation of pyridoxal phosphate enzymes by gabaculine. Correlation with enzymic exchange of beta-protons.

Gabaculine, 5-amino-1,3-cyclohexadienylcarboxylate, is a very efficient enzyme-activated inhibitor of gamma-aminobutyrate transaminase (Rando, R. R. (1977) Biochemistry 16, 4604-4610). However, enzymes for which gamma-aminobutyrate is not a substrate are also inactivated by gabaculine. Thus, purified D-amino acid transaminase, L-alanine transaminase, and L-aspartate transaminase are also inactivated (Ki values of 0.1 mM, 1 mM, and 55 mM, respectively). The effects of this inhibitor on such a diverse group of enzymes appear to be related to the enzymic exchange of beta-protons of their normal substrates. L-Alanine transaminase and L-aspartate transaminase are known to catalyze such an exchange (Walter, U., Luthe, H., Gerhart, F., and Söling, H.-D. (1975) Eur. J. Biochem. 59, 395-403). D-Amino acid transaminase and gamma-aminobutyrate transaminase, which are inactivated by gabaculine, also catalyze exchange of the beta-protons of their substrates. Alanine racemase and tryptophanase, which are known not to catalyze an analogous exchange, were found to be insensitive to gabaculine. We postulate that aromatization of gabaculine, in which the beta-proton is removed, is an enzyme-catalyzed event for those pyridoxal phosphate enzymes that have a nucleophilic group at the active site to catalyze this process.

Synergy in the antimicrobial action of penicillin and beta-chloro-D-alanine in vitro.

beta-Chloro-d-alanine and penicillin G acted on early and late steps, respectively, in the biosynthesis of the bacterial cell wall. In combination these compounds showed a synergistic effect on the growth of Salmonella typhimurium and of Escherichia coli in vitro.

Enzyme-activated inhibition of bacterial D-amino acid transaminase by beta-cyano-D-alanine.

beta-Cyano-D-alanine is an efficient suicide substrate (Ki = 10 microM) of D-amino acid transaminase. This apparent inactivation is temperature dependent: it is irreversible at 10 degrees C or below and becomes progressively reversible at higher temperatures. Since at higher temperatures the apparent reactivation process predominates over the inactivation reaction, the reactivation process is considered to be endothermic. The nature of this reversibility suggests the formation of a heat labile bond between the inhibitor molecule and a nucleophilic group on the enzyme.

Substrate-induced changes in sulfhydryl reactivity of bacterial D-amino acid transaminase.

D-Amino acid transaminase from Bacillus sphaericus strain ATCC 14577 is a dimer with eight cysteinyl residues per molecule (T.S. Soper, W.M. Jones, and J.M. Manning (1979) J. Biol. Chem. 254, 10,901-10,905). The reaction of the cysteinyl residues with a variety of sulfhydryl reagents has been explored to gain insight into the physical environments around these cysteinyl residues in the absence or the presence of substrates. The native enzyme, in the pyridoxal-P conformation, appears to be a symmetrical dimer, whose SH groups react in pairs with anionic reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) or the halo acids. Two SH groups react with either reagent without altering enzymatic activity. Two additional SH groups react with DTNB with loss of catalytic activity. Positively charged reagents such as beta-bromoethylamine are much more effective in inactivating the pyridoxal-P conformation of the enzyme with almost five of the eight SH groups reacting and this results in a significant loss in catalytic activity. The neutral reagent dithiodipyridine is able to detect some asymmetry in the pyridoxal-P conformation. Upon addition of a D-amino acid substrate, the enzyme is transformed into the pyridoxamine-P conformation. This conformation is much more reactive with anionic reagents and much less reactive with cationic reagents, suggesting that there is a significant change in the net charge around one of the SH groups in the pyridoxamine-P conformation. Also, titration with DTNB indicates that the enzyme is a much more asymmetric dimmer in the pyridoxamine-P conformation than in the pyridoxal-P conformation. Thus, upon binding of a D-amino acid substrate, D-amino acid transaminase is transformed into the pyridoxamine-P conformation. This results in a significant change in the environment of four of the sulfhydryl groups of the enzyme. We conclude that the enzyme is transformed from a symmetrical dimer into an asymmetrical dimer and that the net charge of one of the pairs of cysteinyl groups is changed from a net negative charge into a net positive charge. These results suggest that there is a significant conformational change that occurs during the transition from the pyridoxal-P into the pyridoxamine-P form of this transaminase.

Examination of the intersubunit interaction between glutamate-48 and lysine-168 of ribulose-bisphosphate carboxylase/oxygenase by site-directed mutagenesis.

The active site of ribulose-bisphosphate carboxylase/oxygenase is constituted from domains of adjacent subunits and includes an intersubunit electrostatic interaction between Lys 168 and Glu48, which has been recently identified by x-ray crystallography (Andersson, I., Knight, S., Schneider, G., Lindqvist, Y., Lundqvist, T., Brändén, C.-I., and Lorimer, G.H. (1989) Nature 337, 229-234; Lundqvist, T., and Schneider, G. (1989) J. Biol. Chem. 264, 7078-7083). To examine the structural and functional requirements for this interaction, we have used site-directed mutagenesis to replace Lys168 of the homodimeric enzyme from Rhodospirillum rubrum with arginine, glutamine, or glutamic acid. All three substitutions result in mutant enzymes with less than or equal to 0.1% of wild-type activity. The nonconservative substitution of Lys168 with a glutamyl residue precludes the formation of a stable dimer, explaining the consequential abolition of enzymic activity. Both the Arg168 and Gln168 mutant proteins are isolated as stable dimers, even though the latter obviously lacks an electrostatic interaction present in the wild-type enzyme. Despite the absence of overall carboxylase activity, these two mutant proteins serve as catalysts for the enolization of ribulose bisphosphate, as measured by exchange of the C3 proton with solvent. These observations, as well as ligand-binding properties of the mutant proteins, are consistent with Lys168 facilitating a catalytic step subsequent to enolization.

Essentiality of Glu-48 of ribulose bisphosphate carboxylase/oxygenase as demonstrated by site-directed mutagenesis.

Previous reports provide indirect evidence for the presence of Glu-48 at the active site of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. This possibility has been examined directly by replacement of Glu-48 with glutamine via site-directed mutagenesis. This single amino acid substitution does not prevent subunit association or ligand binding. However, the Glu-48 mutant is severely deficient in catalytic activity, exhibiting a kcat only 0.05% that of wild-type enzyme. These results demonstrate that Glu-48 is positioned at the active site and suggest that it serves a functional role. In conjunction with previous studies, the discovery of essentiality of Glu-48 argues that the active site is located at an interface between subunits.

Inactivation of bacterial D-amino acid transaminase by beta-chloro-D-alanine.

Purified D-amino acid transaminase from Bacillus sphaericus catalyzes an alpha,beta elimination from the D isomer of beta-chloroalanine to yield equivalent amounts of pyruvate, chloride, and ammonia; the L isomer of chloroalanine is not a substrate for this transaminase. During the beta elimination there is a synchronous loss in enzyme activity; the Kinact for beta-chloroalanine was estimated to be about 10 micrometers. The alpha-aminoacrylate-Schiff base intermediate formed after beta elimination of chloride ion is probably the key intermediate that partitions between one inactivation event for every 1500 turnovers. In the presence of D-alanine and alpha-ketoglutarate, which are good substrates for the transaminase activity of this enzyme, beta-chloroalanine is a potent, competitive inhibitor (K1 = 10 micrometers) with D-alanine and a weak, uncompetitive inhibitor with alpha-ketoglutarate.

Inactivation of bacterial D-amino acid transaminases by the olefinic amino acid D-vinylglycine.

D-Vinylglycine (2-amino-3-butenoate) functions as a transamination substrate and irreversible inactivator of the homogeneous pyridoxal phosphate-dependent D-amino acid transaminases from Bacillus subtilis and Bacillus sphaericus. In the absence of alpha-ketoglutarate as co-substrate, vinyl-glycine causes little if any inactivation of either enzyme; in the presence of excess alpha-ketoglutarate, both enzymes are inactivated with pseudo-first order kinetics. The limiting rate constant for inactivation of the B. sphaericus enzyme is 1.9 min-1, for the B. subilis enzyme it is 0.36 min-1. The number of catalytic events before inactivation is about 450 for the B. sphaericus enzyme and about 800 for the B. subtilis enzyme; that is, about 0.2% inactivation in each catalytic cycle for the former enzyme and 0.15% for the latter. Comparisons are made with the L-aspartate amino-transferase from pig heart which is inactivated completely in one catalytic cycle and the L-alanine aminotransferase which is not inactivated in many cycles. Comparisons are also made between the likely mode of D-transaminase inactivation produced by vinylglycine and the mode of inactivation induced by beta-chloro-D-alanine.

Essentiality of Lys-329 of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as demonstrated by site-directed mutagenesis.

The unusual chemical properties of active-site Lys-329 of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have suggested that this residue is required for catalysis. To test this postulate Lys-329 was replaced with glycine, serine, alanine, cysteine, arginine, glutamic acid or glutamine by site-directed mutagenesis. These single amino acid substitutions do not appear to induce major conformational changes because (i) intersubunit interactions are unperturbed in that the purified mutant proteins are stable dimers like the wild-type enzyme and (ii) intrasubunit folding is normal in that the mutant proteins bind the competitive inhibitor 6-phosphogluconate with an affinity similar to that of wild-type enzyme. In contrast, all of the mutant proteins are severely deficient in carboxylase activity (less than 0.01% of wild-type) and are unable to form the exchange-inert complex, characteristic of the wild-type enzyme, with the transition-state analogue carboxyarabinitol bisphosphate. These results underscore the stringency of the requirement for a lysyl side-chain at position 329 and imply that Lys-329 is involved in catalysis, perhaps stabilizing a transition state in the overall reaction pathway.

Examination of subunit interactions at the active site of ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum by hybridization of site-directed mutants.

The two active sites of homodimeric ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum are constituted by interacting domains of adjacent subunits, in which residues from each are required for catalytic activity. Active-site residues include Lys-166 of one domain and Glu-48 of the interacting domain from the adjacent subunit. Whereas all substitutions for Lys-166, introduced by site-directed mutagenesis, abolished catalytic activity, only a negatively charged residue (e.g., aspartic acid) resulted in the disruption of the subunit interactions (Lee et al., 1987). This disruption could result from improper folding of the individual polypeptide chains or to more localized effects (e.g., charge-charge repulsion due to proximal negative charges of Asp-166 and Glu-48 of adjacent domains or conformational changes restricted to a single domain). To address these questions, we have examined the ability of the Asp-166 mutant subunit to associate with a mutant subunit in which the negatively charged Glu-48 has been replaced by the neutral glutaminyl residue. Coexpression in Escherichia coli of the genes for both mutant subunits results in formation of a catalytically active hybrid, despite the absence of activity when either gene is expressed individually. Isolation and characterization of the hybrid show that it is composed of one Asp-166 subunit and one Gln-48 subunit, presumably with only one functional active site per dimeric molecule. This association of dissimilar subunits shows that introduction of a negative charge at position 166 does not lead to overall distortion of subunit conformation. In contrast to the wild-type enzyme, the hybrid dissociates spontaneously at low protein concentration but is stabilized by elevated ionic strengths or by glycerol.

Role of asparagine-111 at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as explored by site-directed mutagenesis.

Crystallographic studies of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum suggest that active-site Asn111 interacts with Mg2+ and/or substrate (Lundqvist, T., and Schneider, G. (1991) J. Biol. Chem. 266, 12604-12611). To examine possible catalytic roles of Asn111, we have used site-directed mutagenesis to replace it with a glutaminyl, aspartyl, seryl, or lysyl residue. Although the mutant proteins are devoid of detectable carboxylase activity, their ability to form a quaternary complex comprised of CO2, Mg2+, and a reaction-intermediate analogue is indicative of competence in activation chemistry and substrate binding. The mutant proteins retain enolization activity, as measured by exchange of the C3 proton of ribulose bisphosphate with solvent, thereby demonstrating a preferential role of Asn111 in some later step of overall catalysis. The active sites of this homodimeric enzyme are formed by interactive domains from adjacent subunits (Larimer, F. W., Lee, E. H., Mural, R. J., Soper, T. S., and Hartman, F. C. (1987) J. Biol. Chem. 262, 15327-15329). Crystallography assigns Asn111 to the amino-terminal domain of the active site (Knight, S., Anderson, I., and Brändén, C.-I. (1990) J. Mol. Biol. 215, 113-160). The observed formation of enzymatically active heterodimers by the in vivo hybridization of an inactive position-111 mutant with inactive carboxyl-terminal domain mutants is consistent with this assignment.

Intersubunit location of the active site of ribulose-bisphosphate carboxylase/oxygenase as determined by in vivo hybridization of site-directed mutants.

Ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is a homodimer of 50.5-kDa subunits with two substrate binding sites per molecule of dimer. To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interactions, we have used a novel in vivo approach for producing heterodimers from catalytically inactive, site-directed mutants of the carboxylase. When the alleles encoding these mutant proteins are placed separately into compatible plasmids and coexpressed in the same Escherichia coli host, activity is observed at about 20% of the wild-type level. Analysis of the carboxylase purified from these cells reveals the presence of heterodimers of the two mutant proteins. This interallelic complementation demonstrates that domains from each of the subunits interact to form a shared active site.

Nonessentiality of histidine 291 of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase as determined by site-directed mutagenesis.

Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential active-site residue (Igarashi, Y., McFadden, B. A., and El-Gul, T. (1985) Biochemistry 24, 3957-3962). From the pH dependence of inactivation, the pKa of His-298 was observed to be approximately 6.8, and it was suggested that this histidine might be the essential base that initiates catalysis (Paech, C. (1985) Biochemistry 24, 3194-3199). To explore further the possible function of His-298, we have used site-directed mutagenesis to replace the corresponding residue of the Rhodospirillum rubrum carboxylase (His-291) with alanine. Assays of extracts of Escherichia coli JM107, harboring either the wild-type or mutant gene in an expression vector, revealed that the mutant protein is approximately 40% as active catalytically as the normal carboxylase. After purification to near homogeneity by immunoaffinity chromatography, the mutant protein was partially characterized with respect to subunit structure, kinetic parameters, and interaction with a transition-state analogue. The purified mutant carboxylase had a kcat of 1.5 s-1 and a kcat/Km of 1.7 X 10(4) M-1 s-1 in contrast to values of 3.6 s-1 and 6 X 10(5) M-1 s-1 for the normal enzyme. The high level of enzyme activity exhibited by the Ala-291 mutant excludes His-291 in the R. rubrum carboxylase (and by inference His-298 in the spinach carboxylase) as a catalytically essential residue.

Function of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase as examined by site-directed mutagenesis.

Affinity labeling and comparative sequence analyses have placed Lys-166 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at the active site. The unusual nucleophilicity and acidity of the epsilon-amino group of Lys 166 (pKa = 7.9) suggest its involvement in catalysis, perhaps as the base that enolizes ribulosebisphosphate (Hartman, F.C., Milanez, S., and Lee, E.H. (1985) J. Biol. Chem. 260, 13968-13975). In attempts to clarify the role of Lys-166 of the carboxylase, we have used site-directed mutagenesis to replace this lysyl residue with glycine, alanine, serine, glutamine, arginine, cysteine, or histidine. All seven of these mutant proteins, purified by immunoaffinity chromatography, are severely deficient in carboxylase activity; the serine mutant, which is the most active, has a kcat only 0.2% that of the wild-type enzyme. Although low, the carboxylase activity displayed by some of the mutant proteins proves that Lys-166 is not required for substrate binding and argues that the detrimental effects brought about by amino acid substitutions at position 166 do not reflect gross conformational changes. As demonstrated by their ability to tightly bind a transition-state analogue (2-carboxyarabinitol 1,5-bisphosphate) in the presence of CO2 and Mg2+, some of the mutant proteins undergo the carbamylation reaction that is required for activation of the wild-type enzyme. Since Lys-166 is required neither for activation (i.e. carbamylation by CO2) nor for substrate binding, it must be essential to catalysis. When viewed within the context of previous related studies, the results of site-directed mutagenesis are entirely consistent with Lys-166 functioning as the base that initiates catalysis by abstracting the C-3 proton from ribulosebisphosphate. An alternative possibility that Lys-166 acts to stabilize a transition state in the reaction pathway cannot be rigorously excluded.