Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer

Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis. The Escherichia coli biotin carboxylase is readily isolated from the other components of the acetyl-CoA carboxylase complex such that enzymatic activity is retained. The three-dimensional structure of biotin carboxylase, determined by x-ray crystallography, demonstrated that the enzyme is a homodimer consisting of two active sites in which each subunit contains a complete active site. To understand how each subunit contributes to the overall function of biotin carboxylase, we made hybrid molecules in which one subunit had a wild-type active site, and the other subunit contained an active site mutation known to significantly affect the activity of the enzyme. One of the two genes encoded a poly-histidine tag at its N terminus, whereas the other gene had an N-terminal FLAG epitope tag. The two genes were assembled into a mini-operon that was induced to give high level expression of both enzymes. "Hybrid" dimers composed of one subunit with a wild-type active site and a second subunit having a mutant active site were obtained by sequential chromatographic steps on columns of immobilized nickel chelate and anti-FLAG affinity matrices. In vitro kinetic studies of biotin carboxylase dimers in which both subunits were wild type revealed that the presence of the N-terminal tags did not alter the activity of the enzyme. However, kinetic assays of hybrid dimer biotin carboxylase molecules in which one subunit had an active site mutation (R292A, N290A, K238Q, or E288K) and the other subunit had a wild-type active site resulted in 39-, 28-, 94-, and 285-fold decreases in the activity of these enzymes, respectively. The dominant negative effects of these mutant subunits were also detected in vivo by monitoring the rate of fatty acid biosynthesis by [(14)C]acetate labeling of cellular lipids. Expression of the mutant biotin carboxylase genes from an inducible arabinose promoter resulted in a significantly reduced rate of fatty acid synthesis relative to the same strain that expressed the wild type gene. Thus, both the in vitro and in vivo data indicate that both subunits of biotin carboxylase are required for activity and that the two subunits must be in communication during enzyme function.     

The utility of molecular dynamics simulations for understanding site-directed mutagenesis of glycine residues in biotin carboxylase

Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. Comparison of the crystal structures of biotin carboxylase in the absence and presence of ATP showed a central B-domain closure when ATP was bound. Peptidic NH groups from two active site glycine residues (Gly165 and Gly166) that form hydrogen bonds to the phosphate oxygens of ATP were postulated to act as a "trigger" for movement of the B-domain. The function of these two glycine residues in the catalytic mechanism was studied by disruption of the hydrogen bonds using site-directed mutagenesis. Both single (G165V) and (G166V) and double mutants (G165V-G166V) were constructed. The mutations did not affect the maximal velocity of a partial reaction, the bicarbonate-dependent ATPase activity. This suggests that the peptidic NH groups of Gly165 and Gly166 are not triggers for domain movement. However, the K(m) values for ATP for each of the mutants was increased over 40-fold when compared with wild-type indicating the peptidic NH groups of Gly165 and Gly166 play a role in binding ATP. Consistent with ATP binding, the maximal velocity for the biotin-dependent ATPase activity (i.e. the complete reaction) was decreased over 100-fold suggesting the mutations have misaligned the reactants for optimal catalysis. Molecular dynamics studies confirm perturbation of the hydrogen bonds from the mutated residues to ATP, whereas the double mutant exhibits antagonistic effects such that hydrogen bonding from residues 165 and 166 to ATP is similar to that in the wild-type. Consistent with the site-directed mutagenesis results the molecular dynamics studies show that ATP is misaligned in the mutants.     

The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity

Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5'-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.     

Structural and biochemical studies on the regulation of biotin carboxylase by substrate inhibition and dimerization

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO₂ donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 Å resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca²⁺ ions or two ADP molecules and one Mg²⁺ ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca²⁺ ion and the Mg²⁺ ion are associated with the ADP molecule in the active site, and the other Ca²⁺ ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.     

Modeling and numerical simulation of biotin carboxylase kinetics: implications for half-sites reactivity

Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis in all organisms. In Escherichia coli, biotin carboxylase exists as a homodimer where each subunit contains a complete active site. In a previous study (Janiyani, K., Bordelon, T., Waldrop, G.L., Cronan Jr., J.E., 2001. J. Biol. Chem. 276, 29864-29870), hybrid dimers were constructed where one subunit was wild-type and the other contained an active site mutation that reduced activity at least 100-fold. The activity of the hybrid dimers was only slightly greater than the activity of the mutant homodimers and far less than the expected 50% activity for completely independent active sites. Thus, there is communication between the two subunits of biotin carboxylase. The dominant negative effect of the mutations on the wild-type active site was interpreted as alternating catalytic cycles of the active sites in the homodimer. In order to test the hypothesis of oscillating catalytic cycles, mathematical modeling and numerical simulations of the kinetics of wild-type, hybrid dimers, and mutant homodimers of biotin carboxylase were performed. Numerical simulations of biotin carboxylase kinetics were the most similar to the experimental data when an oscillating active site model was used. In contrast, alternative models where the active sites were independent did not agree with the experimental data. Thus, the numerical simulations of the proposed kinetic model support the hypothesis that the two active sites of biotin carboxylase alternate their catalytic cycles.     

ATPase activity of biotin carboxylase provides evidence for initial activation of HCO3- by ATP in the carboxylation of biotin

When we incubated biotin carboxylase from Escherichia coli with ATP in absence of biotin we observed HCO3- -dependent ATP hydrolysis, which was activated by 10% ethanol in the same proportion as the activity of D-biotin carboxylation assayed in the presence of biotin. The two activities exhibited identical heat stability and were protected equally by glycerol; both required Mg2+ and K+ and showed similar dependency on the concentration of ATP. Biotin assay excluded potential contamination by traces of biotin as a cause of the observed ATP hydrolysis, and this was confirmed by the findings that carboxybiotin did not accumulate and that avidin was uninhibitory. Therefore we concluded that this HCO3- -dependent ATPase was genuinely a partial activity of biotin carboxylase. This partial activity supports a sequential mechanism for enzymatic carboxylation of biotin in which HCO3- is activated by ATP in a first step. It is consistent with the initial formation of the carbonic-phosphoric anhydride (HOCO2PO3(2-)), and it does not agree with models where biotin is phosphorylated by ATP prior to reaction with HCO3-. It appears that enzymes that use HCO3- for carboxylation, including biotin-dependent carboxylases, phosphoenolpyruvate carboxylase, and carbamoyl phosphate synthetase, activate HCO3- by a common mechanism involving the initial formation of the carbonic-phosphoric anhydride.     

Inhibition of biotin carboxylase by a reaction intermediate analog: implications for the kinetic mechanism

The first committed step in long-chain fatty acid synthesis is catalyzed by the multienzyme complex acetyl CoA carboxylase. One component of the acetyl CoA carboxylase complex is biotin carboxylase which catalyzes the ATP-dependent carboxylation of biotin. The Escherichia coli form of biotin carboxylase can be isolated from the other components of the acetyl CoA carboxylase complex such that enzymatic activity is retained. The synthesis of a reaction intermediate analog inhibitor of biotin carboxylase has been described recently (Organic Lett. 1, 99-102, 1999). The inhibitor is formed by coupling phosphonoacetic acid to the 1'-N of biotin. In this paper the characterization of the inhibition of biotin carboxylase by this reaction-intermediate analog is described. The analog showed competitive inhibition versus ATP with a slope inhibition constant of 8 mM. Noncompetitive inhibition was found for the analog versus biotin. Phosphonoacetate exhibited competitive inhibition with respect to ATP and noncompetitive inhibition versus bicarbonate. Biotin was found to be a noncompetitive substrate inhibitor of biotin carboxylase. These data suggested that biotin carboxylase had an ordered addition of substrates with ATP binding first followed by bicarbonate and then biotin.     

The biotin domain peptide from the biotin carboxyl carrier protein of Escherichia coli acetyl-CoA carboxylase causes a marked increase in the catalytic efficiency of biotin carboxylase and carboxyltransferase relative to free biotin.

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase activity, a biotin carboxyl carrier protein, and a carboxyltransferase activity. The C-terminal 87 amino acids of the biotin carboxyl carrier protein (BCCP87) form a domain that can be independently expressed, biotinylated, and purified (Chapman-Smith, A., Turner, D. L., Cronan, J. E., Morris, T. W., and Wallace, J. C. (1994) Biochem. J. 302, 881-887). The ability of the biotinylated form of this 87-residue protein (holoBCCP87) to act as a substrate for biotin carboxylase and carboxyltransferase was assessed and compared with the results with free biotin. In the case of biotin carboxylase holoBCCP87 was an excellent substrate with a K(m) of 0.16 +/- 0.05 mM and V(max) of 1000.8 +/- 182.0 min(-1). The V/K or catalytic efficiency of biotin carboxylase with holoBCCP87 as substrate was 8000-fold greater than with biotin as substrate. Stimulation of the ATP synthesis reaction of biotin carboxylase where carbamyl phosphate reacted with ADP by holoBCCP87 was 5-fold greater than with an equivalent amount of biotin. The interaction of holoBCCP87 with carboxyltransferase was characterized in the reverse direction where malonyl-CoA reacted with holoBCCP87 to form acetyl-CoA and carboxyholoBCCP87. The K(m) for holoBCCP87 was 0.45 +/- 0.07 mM while the V(max) was 2031.8 +/- 231.0 min(-1). The V/K or catalytic efficiency of carboxyltransferase with holoBCCP87 as substrate is 2000-fold greater than with biotin as substrate.     

Catalytic mechanism of biotin carboxylase: steady-state kinetic investigations

Biotin carboxylase was purified from Escherichia coli by a new procedure, and its steady-state kinetic parameters were examined. MgATP and bicarbonate add to the enzyme randomly, followed by addition of biotin. Both bicarbonate and MgATP add in rapid equilibrium. A catalytic base with a pK of 6.6 is observed in V/K profiles. Inactivation studies also revealed a sulfhydryl group in the active site that is essential for catalysis. It is proposed that the acid-base catalysts are necessary for the tautomerization of biotin, which presumably enhances its nucleophilicity toward the carboxyl group donor. A second enzymic group with a pK of 6.6, whose role is unknown, is seen in Vmax profiles. The pH profiles for the biotin carboxylase catalyzed phosphorylation of ADP by carbamoyl phosphate have the same shape as the profiles for the forward reaction, which demonstrates that the enzymic bases assume the same protonation states for catalysis of transphosphorylation in either direction. The lack of reactivity of thionucleotide analogues of ATP when Mg is used as the divalent metal ion suggests that both metal ions required for reaction coordinate to the nucleotide. The second metal ion appears to be absolutely required for reaction and not merely an activator of the reaction. Characterization of a bicabonate-dependent biotin-independent ATPase activity strongly suggests that carboxylation proceeds via a carboxyphosphate intermediate.     

On the intermediacy of carboxyphosphate in biotin-dependent carboxylations

In the ATP-dependent carboxylation of biotin that is catalyzed by most biotin-dependent carboxylases, a fundamental mechanistic question is whether the ATP activates bicarbonate (via the formation of carboxyphosphate as an intermediate) or whether the ATP activates biotin (via the formation of O-phosphobiotin). We have resorted to three mechanistic tests using the biotin carboxylase subunit of acetyl-CoA carboxylase from Escherichia coli: positional isotope exchange, intermediate trapping, and 18O tracer experiments on the ATPase activity. First, no catalysis of positional isotope exchange in adenosine 5'-[( alpha, beta-18O, beta, beta-18O2]triphosphate) was observed when either biotin or bicarbonate was absent, nor was any exchange seen in the presence of both N-1-methylbiotin and bicarbonate. Second, the putative carboxyphosphate intermediate could not be trapped as its trimethyl ester, under conditions of incubation and analysis where the authentic triester was shown to be adequately stable. In the third test, however, we showed that the ATPase activity of biotin carboxylase that is seen in the absence of biotin, an activity that is known to parallel the normal carboxylase reaction when biotin is present, occurs with the transfer of an 18O label directly from [18O]bicarbonate into the product Pi. This result suggests that the bicarbonate-dependent biotin-independent ATPase reaction catalyzed by biotin carboxylase goes via carboxyphosphate and that the carboxylation of biotin itself may proceed analogously.     

Movement of the biotin carboxylase B-domain as a result of ATP binding

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In Escherichia coli, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. The biotin carboxylase component has served for many years as a paradigm for mechanistic studies devoted toward understanding more complicated biotin-dependent carboxylases. The three-dimensional x-ray structure of an unliganded form of E. coli biotin carboxylase was originally solved in 1994 to 2.4-A resolution. This study revealed the architecture of the enzyme and demonstrated that the protein belongs to the ATP-grasp superfamily. Here we describe the three-dimensional structure of the E. coli biotin carboxylase complexed with ATP and determined to 2.5-A resolution. The major conformational change that occurs upon nucleotide binding is a rotation of approximately 45(o) of one domain relative to the other domains thereby closing off the active site pocket. Key residues involved in binding the nucleotide to the protein include Lys-116, His-236, and Glu-201. The backbone amide groups of Gly-165 and Gly-166 participate in hydrogen bonding interactions with the phosphoryl oxygens of the nucleotide. A comparison of this closed form of biotin carboxylase with carbamoyl-phosphate synthetase is presented.     

Kinetic characterization of mutations found in propionic acidemia and methylcrotonylglycinuria: evidence for cooperativity in biotin carboxylase

Acetyl-CoA carboxylase catalyzes the committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multifunctional enzyme consisting of three separate proteins: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source, has a homologous functionally identical subunit in the mammalian biotin-dependent enzymes propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase. In humans, mutations in either of these enzymes result in the metabolic deficiency propionic acidemia or methylcrotonylglycinuria. The lack of a system for structure-function studies of these two biotin-dependent carboxylases has prevented a detailed analysis of the disease-causing mutations. However, structural data are available for E. coli biotin carboxylase as is a system for its overexpression and purification. Thus, we have constructed three site-directed mutants of biotin carboxylase that are homologous to three missense mutations found in propionic acidemia or methylcrotonylglycinuria patients. The mutants M169K, R338Q, and R338S of E. coli biotin carboxylase were selected for study to mimic the disease-causing mutations M204K and R374Q of propionyl-CoA carboxylase and R385S of 3-methylcrotonyl-CoA carboxylase. These three mutants were subjected to a rigorous kinetic analysis to determine the function of the residues in the catalytic mechanism of biotin carboxylase as well as to establish a molecular basis for the two diseases. The results of the kinetic studies have revealed the first evidence for negative cooperativity with respect to bicarbonate and suggest that Arg-338 serves to orient the carboxyphosphate intermediate for optimal carboxylation of biotin.     

Site-directed mutagenesis of the conserved histidine residue of phosphoenolpyruvate carboxylase. His138 is essential for the second partial reaction.

Histidine residues have previously been suggested to be essential for the activity of phosphoenolpyruvate carboxylase as demonstrated by chemical modification of these residues. Although the location of these residues on the primary structure is not known, a comparison of nine phosphoenolpyruvate (P-pyruvate) carboxylases sequenced recently revealed that there are only two conserved histidine residues (His138 and His579, coordinates from the E. coli enzyme). Site-directed mutagenesis of these residues were undertaken with the E. coli P-pyruvate carboxylase and the properties of purified mutant enzymes were investigated. Mutation of His138 to asparagine (H138N) produced a protein which did not show carboxylase activity. However, this mutant enzyme catalyzed the bicarbonate-dependent dephosphorylation (Vmax = 1.4 mumol.min-1.mg-1) of the P-pyruvate. Since this reaction is due to one of the two partial reactions proposed for this enzyme, the results indicate that His138 is obligatory for the second-step reaction, i.e. the carboxylation of the enolate form of pyruvate by carboxyphosphate. Mutation of His579 to asparagine (H579N) produced an enzyme which had 69% of the wild-type carboxylase activity, but its affinity for P-pyruvate was decreased by 24-fold.     

Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme

Pyruvate carboxylase is a biotin-dependent enzyme in which the biotin is carboxylated by a putative carboxyphosphate intermediate that is formed in a reaction between ATP and bicarbonate. The resultant carboxybiotin then transfers its carboxyl group to pyruvate to form oxaloacetate. In the Bacillus thermodenitrificans enzyme the biotin is covalently attached to K1112. A mutant form of the enzyme (K1112A) has been prepared which is not biotinylated. This mutant did not catalyse the complete reaction, but did catalyse ATP-cleavage and the carboxylation of free biotin. Oxaloacetate decarboxylation was not catalysed, even in the presence of free biotin, suggesting that only the biotin carboxylation domain of the enzyme is accessible to free biotin. This mutant allowed the study of ATP-cleavage both coupled and not coupled to biotin carboxylation. Kinetic analyses of these reactions indicate that the major effect of the enzyme activator, acetyl CoA, is to promote the carboxylation of biotin. Acetyl CoA reduces the K(m)s for both MgATP and biotin. In addition, pH profiles of the ATP-cleavage reaction in the presence and absence of free biotin revealed the involvement of several ionisable residues in both ATP-cleavage and biotin carboxylation. K1112A also catalyses the phosphorylation of ADP from carbamoyl phosphate. Stopped-flow studies using the fluorescent ATP analogue, formycin A-5'-triphosphate, in which nucleotide binding to the holoenzyme was compared to K1112A indicated that the presence of biotin enhanced binding. Attempts to trap the putative carboxyphosphate intermediate in K1112A using diazomethane were unsuccessful.     

The birA gene of Escherichia coli encodes a biotin holoenzyme synthetase

Mutations in the birA gene of Escherichia coli cause defects in biotin operon repression, biotin uptake and retention of intracellular biotin (Campbell et al., 1972: Barker &, Campbell, 1980). We report here that the birA gene encodes the major biotin-fixing enzyme of this organism, the acetyl-CoA carboxylase biotin holoenzyme synthetase (EC 6.3.4.15). Unlike the situation in wild-type E. coli extracts, measurements of labeled biotin incorporation into protein in sonicated extracts reveal no in vitro activity. Three different mutants exhibit altered holoenzyme synthetase activity, including one clear instance of a thermolabile activity specified by birA361.Amplification of birA gene expression by infection of cells with a λ phage bearing an EcoRI fragment of the E. coli chromosome which includes the gene results in a 20- to 40-fold increase in specific activity. When the λbirA phage carries the birA85 mutation, no activity increase is observed. Infection of cells with a λbirA361 transducing phage results in a 20- to 40-fold increase in temperature-sensitive activity. We have purified the activity specified by birA361 approximately 1000-fold and have shown that the purified enzyme is more thermolabile than similarly purified wild-type enzyme.Measurements of holoenzyme synthetase in extracts and biotin uptake by whole cells indicate that certain mutations located at the same chromosomal position as birA mutations but initially characterized as defective only in bio repression are also deficient in biotin holoenzyme synthetase and biotin uptake. This result indicates that all mutations at this location affect the same enzyme, and we have redesignated these “bioR” mutations as birA. Results of complementation analysis of birA mutations and biochemical characterization of the gene and its product, presented in the accompanying paper, support the view that the birA product functions both as the bio repressor and biotin holoenzyme synthetase.     

Dimerization of the bacterial biotin carboxylase subunit is required for acetyl coenzyme A carboxylase activity in vivo

Acetyl coenzyme A (acteyl-CoA) carboxylase (ACC) is the first committed enzyme of the fatty acid synthesis pathway. Escherichia coli ACC is composed of four different proteins. The first enzymatic activity of the ACC complex, biotin carboxylase (BC), catalyzes the carboxylation of the protein-bound biotin moiety of another subunit with bicarbonate in an ATP-dependent reaction. Although BC is found as a dimer in cell extracts and the carboxylase activities of the two subunits of the dimer are interdependent, mutant BC proteins deficient in dimerization are reported to retain appreciable activity in vitro (Y. Shen, C. Y. Chou, G. G. Chang, and L. Tong, Mol. Cell 22:807-818, 2006). However, in vivo BC must interact with the other proteins of the complex, and thus studies of the isolated BC may not reflect the intracellular function of the enzyme. We have tested the abilities of three BC mutant proteins deficient in dimerization to support growth and report that the two BC proteins most deficient in dimerization fail to support growth unless expressed at high levels. In contrast, the wild-type protein supports growth at low expression levels. We conclude that BC must be dimeric to fulfill its physiological function.     

Functional interaction of Azotobacter vinelandii cytoplasmic cyclophilin with the biotin carboxylase subunit of acetyl-CoA carboxylase

Cyclophilins (E.C. 5.1.2.8) are protein chaperones with peptidyl-prolyl cis/trans isomerase activity (PPIase). In the present study, we demonstrate a physical interaction among AvppiB, encoding the cytoplasmic cyclophilin from the soil nitrogen-fixing bacterium Azotobacter vinelandii, and AvaccC, encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. A decrease in AvppiB PPIase activity, in the presence of AvaccC, further confirms the interaction. However, PPIase activity seems not to be essential for these interactions since a PPIase active site mutant of cyclophilin does not abolish the AvaccC binding. We further show that the presence of cyclophilin largely influences the measured ATP hydrolyzing activity of AvaccA in a way that is negatively regulated by the PPIase activity. Taken together, our data support a novel role for cyclophilin in regulating biotin carboxylase activity.     

Effect of Ssc protein mutations on the outer membrane permeability barrier function in Salmonella typhimurium: a study using ssc mutant alleles made by site-directed mutagenesis

We have previously discovered and characterized a novel essential enterobacterial protein, the Ssc protein of Salmonella typhimurium and found that the mutation Val291----Met in this protein inhibits bacterial growth at 42 degrees C and the function of its outer membrane permeability barrier at 37 degrees C [7]. In the present paper we prepared, by site-directed mutagenesis, a series of novel plasmid-encoded Ssc mutant proteins and tested their ability to compensate the loss of wild-type Ssc. The mutant proteins Met288----Lys and Gly289----Asp completely lacked this ability, and accordingly, were very defective. Ssc mutants Met288----Leu, Met290----Lys, and Met292----Lys were partially defective. Mutants Met290----Leu and Met292----Leu were non-defective as were also four randomly made mutant proteins with mutations outside the 288-292 region. The S. typhimurium derivative which contained both the chromosomally encoded Ssc Val291----Met and the plasmid-encoded Ssc Gly289----Asp had an outer membrane defect more severe than that caused by SscMet291 only. The mutant Ssc proteins had very little, if any, effect on the outer membrane function in the presence of wild-type Ssc. Even though the function of Ssc is not yet known, our results indicate that region 288-292 is important and that SscAsp289 is thus far the most defective mutant Ssc.     

Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains

Biotinylation in vivo is an extremely selective post-translational event where the enzyme biotin protein ligase (BPL) catalyzes the covalent attachment of biotin to one specific and conserved lysine residue of biotin-dependent enzymes. The biotin-accepting lysine, present in a conserved Met-Lys-Met motif, resides in a structured domain that functions as the BPL substrate. We have employed phage display coupled with a genetic selection to identify determinants of the biotin domain (yPC-104) of yeast pyruvate carboxylase 1 (residues 1075-1178) required for interaction with BPL. Mutants isolated using this strategy were analyzed by in vivo biotinylation assays performed at both 30 degrees C and 37 degrees C. The temperature-sensitive substrates were reasoned to have structural mutations, leading to compromised conformations at the higher temperature. This interpretation was supplemented by molecular modeling of yPC-104, since these mutants mapped to residues involved in defining the structure of the biotin domain. In contrast, substitution of the Met residue N-terminal to the target lysine with either Val or Thr produced mutations that were temperature-insensitive in the in vivo assay. Furthermore, these two mutant proteins and wild-type yPC-104 showed identical susceptibility to trypsin, consistent with these substitutions having no structural effect. Kinetic analysis of enzymatic biotinylation using purified Met --> Thr/Val mutant proteins with both yeast and Escherichia coli BPLs revealed that these substitutions had a strong effect upon K(m) values but not k(cat). The Met --> Thr mutant was a poor substrate for both BPLs, whereas the Met --> Val substitution was a poor substrate for bacterial BPL but had only a 2-fold lower affinity for yeast BPL than the wild-type peptide. Our data suggest that substitution of Thr or Val for the Met N-terminal of the biotinyl-Lys results in mutants specifically compromised in their interaction with BPL.