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.     

PurT-encoded glycinamide ribonucleotide transformylase. Accommodation of adenosine nucleotide analogs within the active site

PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, functions in purine biosynthesis by catalyzing the formylation of glycinamide ribonucleotide through a catalytic mechanism requiring Mg(2+)ATP and formate. From previous x-ray diffraction analyses, it has been demonstrated that PurT transformylase from Escherichia coli belongs to the ATP-grasp superfamily of enzymes, which are characterized by three structural motifs referred to as the A-, B-, and C-domains. In all of the ATP-grasp enzymes studied to date, the adenosine nucleotide ligands are invariably wedged between the B- and C-domains, and in some cases, such as biotin carboxylase and carbamoyl phosphate synthetase, the B-domains move significantly upon nucleotide binding. Here we present a systematic and high-resolution structural investigation of PurT transformylase complexed with various adenosine nucleotides or nucleotide analogs including Mg(2+)ATP, Mg(2+)-5'-adenylylimidodiphosphate, Mg(2+)-beta,gamma-methyleneadenosine 5'-triphosphate, Mg(2+)ATPgammaS, or Mg(2+)ADP. Taken together, these studies indicate that the conformation of the so-called "T-loop," delineated by Lys-155 to Gln-165, is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket. This sensitivity to nucleotide identity is in sharp contrast to that observed for the "P-loop"-containing enzymes, in which the conformation of the binding motif is virtually unchanged in the presence or absence of nucleotides.     

The ATP-grasp enzymes

The ATP-grasp enzymes consist of a superfamily of 21 proteins that contain an atypical ATP-binding site, called the ATP-grasp fold. The ATP-grasp fold is comprised of two α+β domains that "grasp" a molecule of ATP between them and members of the family typically have an overall structural design containing three common conserved focal domains. The founding members of the family consist of biotin carboxylase, d-ala-d-ala ligase and glutathione synthetase, all of which catalyze the ATP-assisted reaction of a carboxylic acid with a nucleophile via the formation of an acylphosphate intermediate. While most members of the superfamily follow this mechanistic pathway, studies have demonstrated that two enzymes catalyze only the phosphoryl transfer step and thus are kinases instead of ligases. Members of the ATP-grasp superfamily are found in several metabolic pathways including de novo purine biosynthesis, gluconeogenesis, and fatty acid synthesis. Given the critical nature of these enzymes, researchers have actively sought the development of potent inhibitors of several members of the superfamily as antibacterial and anti-obseity agents. In this review, we will discuss the structure, function, mechanism, and inhibition of the ATP-grasp enzymes.     

A diverse superfamily of enzymes with ATP-dependent carboxylate-amine/thiol ligase activity.

The recently developed PSI-BLAST method for sequence database search and methods for motif analysis were used to define and expand a superfamily of enzymes with an unusual nucleotide-binding fold, referred to as palmate, or ATP-grasp fold. In addition to D-alanine-D-alanine ligase, glutathione synthetase, biotin carboxylase, and carbamoyl phosphate synthetase, enzymes with known three-dimensional structures, the ATP-grasp domain is predicted in the ribosomal protein S6 modification enzyme (RimK), urea amidolyase, tubulin-tyrosine ligase, and three enzymes of purine biosynthesis. All these enzymes possess ATP-dependent carboxylate-amine ligase activity, and their catalytic mechanisms are likely to include acylphosphate intermediates. The ATP-grasp superfamily also includes succinate-CoA ligase (both ADP-forming and GDP-forming variants), malate-CoA ligase, and ATP-citrate lyase, enzymes with a carboxylate-thiol ligase activity, and several uncharacterized proteins. These findings significantly extend the variety of the substrates of ATP-grasp enzymes and the range of biochemical pathways in which they are involved, and demonstrate the complementarity between structural comparison and powerful methods for sequence analysis.     

Function of conserved residues of human glutathione synthetase: implications for the ATP-grasp enzymes

Glutathione synthetase is an enzyme that belongs to the glutathione synthetase ATP-binding domain-like superfamily. It catalyzes the second step in the biosynthesis of glutathione from gamma-glutamylcysteine and glycine in an ATP-dependent manner. Glutathione synthetase has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. A variety of structural alignment methods were applied and four highly conserved residues of human glutathione synthetase (Glu-144, Asn-146, Lys-305, and Lys-364) were identified in the binding site. The function of these was studied by experimental and computational site-directed mutagenesis. The three-dimensional coordinates for several human glutathione synthetase mutant enzymes were obtained using molecular mechanics and molecular dynamics simulation techniques, starting from the reported crystal structure of human glutathione synthetase. Consistent with circular dichroism spectroscopy, our results showed no major changes to overall enzyme structure upon residue mutation. However, semiempirical calculations revealed that ligand binding is affected by these mutations. The key interactions between conserved residues and ligands were detected and found to be essential for enzymatic activity. Particularly, the negatively charged Glu-144 residue plays a major role in catalysis.     

Molecular structure of Escherichia coli PurT-encoded glycinamide ribonucleotide transformylase

In Escherichia coli, the PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, catalyzes an alternative formylation of glycinamide ribonucleotide (GAR) in the de novo pathway for purine biosynthesis. On the basis of amino acid sequence analyses, it is known that the PurT transformylase belongs to the ATP-grasp superfamily of proteins. The common theme among members of this superfamily is a catalytic reaction mechanism that requires ATP and proceeds through an acyl phosphate intermediate. All of the enzymes belonging to the ATP-grasp superfamily are composed of three structural motifs, termed the A-, B-, and C-domains, and in each case, the ATP is wedged between the B- and C-domains. Here we describe two high-resolution X-ray crystallographic structures of PurT transformylase from E. coli: one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the second with bound AMPPNP and GAR. The latter structure is of special significance because it represents the first ternary complex to be determined for a member of the ATP-grasp superfamily involved in purine biosynthesis and as such provides new information about the active site region involved in ribonucleotide binding. Specifically in PurT transformylase, the GAR substrate is anchored to the protein via Glu 82, Asp 286, Lys 355, Arg 362, and Arg 363. Key amino acid side chains involved in binding the AMPPNP to the enzyme include Arg 114, Lys 155, Glu 195, Glu 203, and Glu 267. Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 A from one of the gamma-phosphoryl oxygens of the AMPPNP.     

Three-dimensional structure of N5-carboxyaminoimidazole ribonucleotide synthetase: a member of the ATP grasp protein superfamily

Escherichia coli PurK, a dimeric N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase, catalyzes the conversion of 5-aminoimidazole ribonucleotide (AIR), ATP, and bicarbonate to N5-CAIR, ADP, and Pi. Crystallization of both a sulfate-liganded and the MgADP-liganded E. coli PurK has resulted in structures at 2.1 and 2.5 A resolution, respectively. PurK belongs to the ATP grasp superfamily of C-N ligase enzymes. Each subunit of PurK is composed of three domains (A, B, and C). The B domain contains a flexible, glycine-rich loop (B loop, T123-G130) that is disordered in the sulfate-PurK structure and becomes ordered in the MgADP-PurK structure. MgADP is wedged between the B and C domains, as with all members of the ATP grasp superfamily. Other enzymes in this superfamily contain a conserved Omega loop proposed to interact with the B loop, define the specificity of their nonnucleotide substrate, and protect the acyl phosphate intermediate formed from this substrate. PurK contains a minimal Omega loop without conserved residues. In the reaction catalyzed by PurK, carboxyphosphate is the putative acyl phosphate intermediate. The sulfate of the sulfate ion-liganded PurK interacts electrostatically with Arg 242 and the backbone amide group of Asn 245, components of the J loop of the C domain. This sulfate may reveal the location of the carboxyphosphate binding site. Conserved residues within the C-terminus of the C domain define a pocket that is proposed to bind AIR in collaboration with an N-terminal strand loop helix motif in the A domain (P loop, G8-L1). The P loop is proposed to bind the phosphate of AIR on the basis of similar binding sites observed in PurN and PurE and proposed in PurD and PurT, four other enzymes in the purine pathway.     

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.     

Gamma-glutamylcysteine synthetase-glutathione synthetase: domain structure and identification of residues important in substrate and glutathione binding

In most organisms, glutathione (GSH) is synthesized by the sequential action of distinct enzymes, gamma-glutamylcysteine synthetase (gamma-GCS) and GSH synthetase (GS). In Streptococcus agalactiae, GSH synthesis is catalyzed by a single enzyme, gamma-glutamylcysteine synthetase-glutathione synthetase (gamma-GCS-GS). The N-terminal sequence of gamma-GCS-GS is similar to Escherichia coli gamma-GCS, but the C-terminal sequence is an ATP-grasp domain more similar to d-Ala, d-Ala ligase than to any known GS. In the present studies, C-terminally and N-terminally truncated constructs were characterized in order to define the limits of the gamma-GCS and GS domains, respectively. Although WT gamma-GCS-GS is nearly uninhibited by GSH (K(i) approximately 140 mM), shorter gamma-GCS domain constructs were unexpectedly found to be strongly inhibited (K(i) approximately 15 mM), reproducing a physiologically important regulation seen in monofunctional gamma-GCS enzymes. Because studies with E. coli gamma-GCS implicate a flexible loop region in GSH binding, chimeras of S. agalactiae gamma-GCS-GS were made containing gamma-GCS domain flexible loop sequences from Enterococcus faecalis and Pasteurella multocida gamma-GCS-GS, isoforms that are inhibited by GSH. Inhibition remained S. agalactiae-like (i.e., very weak). C-Terminal constructs of gamma-GCS-GS have GS activity (0.01-0.04% of WT), but proper folding and significant GS activity required a covalently linked gamma-GCS domain. In addition, site-directed mutants in the middle region of the gamma-GCS-GS sequence established that GS activity depends on residues in a region that is also part of the gamma-GCS domain. Our results provide new insights into the structure of gamma-GCS-GS and suggest gamma-GCS-GS evolved from a monomeric gamma-GCS that became C-terminally fused to a multimeric ATP-grasp protein.     

Crystal structure of urea carboxylase provides insights into the carboxyltransfer reaction

Urea carboxylase (UC) is conserved in many bacteria, algae, and fungi and catalyzes the conversion of urea to allophanate, an essential step in the utilization of urea as a nitrogen source in these organisms. UC belongs to the biotin-dependent carboxylase superfamily and shares the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains with these other enzymes, but its carboxyltransferase (CT) domain is distinct. Currently, there is no information on the molecular basis of catalysis by UC. We report here the crystal structure of the Kluyveromyces lactis UC and biochemical studies to assess the structural information. Structural and sequence analyses indicate the CT domain of UC belongs to a large family of proteins with diverse functions, including the Bacillus subtilis KipA-KipI complex, which has important functions in sporulation regulation. A structure of the KipA-KipI complex is not currently available, and our structure provides a framework to understand the function of this complex. Most interestingly, in the structure the CT domain interacts with the BCCP domain, with biotin and a urea molecule bound at its active site. This structural information and our follow-up biochemical experiments provided molecular insights into the UC carboxyltransfer reaction. Several structural elements important for the UC carboxyltransfer reaction are found in other biotin-dependent carboxylases and might be conserved within this family, and our data could shed light on the mechanism of catalysis of these enzymes.     

A Substrate-induced Biotin Binding Pocket in the Carboxyltransferase Domain of Pyruvate Carboxylase

Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp⁵⁹⁰ and Tyr⁶²⁸ and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.     

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.     

Characterization of the acyl substrate binding pocket of acetyl-CoA synthetase

AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to acetyl-CoA in a two-step reaction. This enzyme is a member of the adenylate-forming enzyme superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- and aryl-CoA synthetases/ligases. Although the structures of several superfamily members demonstrate that these enzymes have a similar fold and domain structure, the low sequence conservation and diversity of the substrates utilized have limited the utility of these structures in understanding substrate binding in more distantly related enzymes in this superfamily. The crystal structures of the Salmonella enterica ACS and Saccharomyces cerevisiae ACS1 have allowed a directed approach to investigating substrate binding and catalysis in ACS. In the S. enterica ACS structure, the propyl group of adenosine 5'-propylphosphate, which mimics the acyl-adenylate intermediate, lies in a hydrophobic pocket. Modeling of the Methanothermobacter thermautotrophicus Z245 ACS (MT-ACS1) on the S. cerevisiae ACS structure showed similar active site architecture, and alignment of the amino acid sequences of proven ACSs indicates that the four residues that compose the putative acetate binding pocket are well conserved. These four residues, Ile312, Thr313, Val388, and Trp416 of MT-ACS1, were targeted for alteration, and our results support that they do indeed form the acetate binding pocket and that alterations at these positions significantly alter the enzyme's affinity for acetate as well as the range of acyl substrates that can be utilized. In particular, Trp416 appears to be the primary determinant for acyl chain length that can be accommodated in the binding site.     

Mutations at four active site residues of biotin carboxylase abolish substrate-induced synergism by 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 protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report a system for site-directed mutagenesis of the biotin carboxylase component is described. The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid. Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography. This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase. In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function. The mutations did not affect the bicarbonate-dependent ATPase reaction. In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin. The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations. The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes. The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme. Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase.     

Identification of active site residues in Bradyrhizobium japonicum acetyl-CoA synthetase.

Acetyl-CoA synthetase (ACS) catalyses the activation of acetate to acetyl-CoA in the presence of ATP and CoA. The gene encoding Bradyrhyzobium japonicum ACS has been cloned, sequenced, and expressed in Escherichia coli. The enzyme comprises 648 amino acid residues with a calculated molecular mass of 71,996 Da. The recombinant enzyme was also purified from the transformed E. coli. The enzyme was essentially indistinguishable from the ACS of B. japonicum bacteroids as to the criteria of polyacrylamide gel electrophoresis and biochemical properties. Based on the results of database analysis, Gly-263, Gly-266, Lys-269, and Glu-414 were selected for site-directed mutagenesis in order to identify amino acid residues essential for substrate binding and/or catalysis. Four different mutant enzymes (G263I, G266I, K269G, and E414Q) were prepared and then subjected to steady-state kinetic studies. The kinetic data obtained for the mutants suggest that Gly-266 and Lys-269 participate in the formation of acetyl-AMP, whereas Glu-414 may play a role in acetate binding.     

Crystal structure of carbapenam synthetase (CarA)

Carbapenam synthetase (CarA) is an ATP/Mg2+-dependent enzyme that catalyzes formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis. CarA is homologous to beta-lactam synthetase (beta-LS), which is involved in clavulanic acid biosynthesis. The catalytic cycles of CarA and beta-LS mediate substrate adenylation followed by beta-lactamization via a tetrahedral intermediate or transition state. Another member of this family of ATP/Mg2+-dependent enzymes, asparagine synthetase (AS-B), catalyzes intermolecular, rather than intramolecular, amide bond formation in asparagine biosynthesis. The crystal structures of apo-CarA and CarA complexed with the substrate (2S,5S)-5-carboxymethylproline (CMPr), ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP), and a single Mg2+ ion have been determined. CarA forms a tetramer. Each monomer resembles beta-LS and AS-B in overall fold, but key differences are observed. The N-terminal domain lacks the glutaminase active site found in AS-B, and an extended loop region not observed in beta-LS or AS-B is present. Comparison of the C-terminal synthetase active site to that in beta-LS reveals that the ATP binding site is highly conserved. By contrast, variations in the substrate binding pocket reflect the different substrates of the two enzymes. The Mg2+ coordination is also different. Several key residues in the active site are conserved between CarA and beta-LS, supporting proposed roles in beta-lactam formation. These data provide further insight into the structures of this class of enzymes and suggest that CarA might be a versatile target for protein engineering experiments aimed at developing improved production methods and new carbapenem antibiotics.     

Effect of mutations at Met-88 and Met-90 on the biotination of Lys-89 of the apo 1.3S subunit of transcarboxylase

The apo 1.3S subunit of transcarboxylase contains the sequence Ala-87-Met-88-Lys-89-Met-90, and it is Lys-89 that is biotinated. This sequence is highly conserved in all the biotin enzymes that have been sequenced (with the exception of acetyl-CoA carboxylase from chicken liver, which has Val in place of Ala). The role of Met-88 and Met-90 in specifying Lys-89 for biotination by synthetase was examined by site-directed mutagenesis. Genes of the 1.3S subunit coding for Thr-88, Leu-88, or Leu-90 were generated by oligonucleotide-directed in vitro mutagenesis and expressed in Escherichia coli. The mutated apo 1.3S subunits were isolated and the biotination by homogeneous synthetase from Propionibacterium shermanii was compared with that of the apo wild-type subunit. The Vmax for the apo mutants was the same as that for the apo wild type, but when Leu was substituted for Met-88 or Met-90, the Km for the mutant was lower than that of the wild-type or mutant Thr-88. The activity of the synthetase of E. coli was determined by an in vivo assay. During the early log phase of growth, a smaller portion of mutants Thr-88 and Leu-90 was biotinated than with the wild-type or mutant Leu-88. When the cultures progressed to stationary phase, mutants and the wild type were biotinated to the same extent. The overall results show that Met-88 and Met-90 are not required for biotination of the apo 1.3S subunit by the synthetases.     

Crystal structure of a lysine biosynthesis enzyme,LysX, from Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the alpha-aminoadipate pathway, which uses alpha-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of alpha-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0A and 2.38A resolutions, respectively. LysX is composed of three alpha+beta domains, each composed of a four to five-stranded beta-sheet core flanked by alpha-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as D-Ala:D-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity.     

Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or "flip-flop" their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF(2)P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC.     

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.