A Novel Signal Transduction Protein PII Variant from Synechococcus elongatus PCC 7942 Indicates a Two-Step Process for NAGK-PII Complex Formation

P_II signal transduction proteins are highly conserved in bacteria, archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC 7942, P_II binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP binding sites also accepting ADP. Depending on its effector molecule binding status, P_II (from this cyanobacterium and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway, N-acetyl-l-glutamate kinase (NAGK), to control arginine biosynthesis. To gain deeper insights into the process of P_II binding to NAGK, we searched for P_II variants with altered binding characteristics and found P_II variants I86N and I86T to be able to bind to an NAGK variant (R233A) that was previously shown to be unable to bind wild-type P_II protein. Analysis of interactions between these P_II variants and wild-type NAGK as well as with the NAGK R233A variant suggested that the P_II I86N variant was a superactive NAGK binder. To reveal the structural basis of this property, we solved the crystal structure of the P_II I86N variant at atomic resolution. The large T-loop, which prevails in most receptor interactions of P_II proteins, is present in a tightly bended conformation that mimics the T-loop of S. elongatus P_II after having latched onto NAGK. Moreover, both P_II I86 variants display a specific defect in 2-OG binding, implying a role of residue I86 in 2-OG binding. We propose a two-step model for the mechanism of P_II-NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of P_II initiates the bending of the extended T-loop of P_II, followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex.     

Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor

N-acetyl-l-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K_m for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity.     

Structural bases of feed-back control of arginine biosynthesis, revealed by the structures of two hexameric N-acetylglutamate kinases, from Thermotoga maritima and Pseudomonas aeruginosa

N-Acetylglutamate kinase (NAGK) catalyses the second step in the route of arginine biosynthesis. In many organisms this enzyme is inhibited by the final product of the route, arginine, and thus plays a central regulatory role. In addition, in photosynthetic organisms NAGK is the target of the nitrogen-signalling protein PII. The 3-D structure of homodimeric, arginine-insensitive, Escherichia coli NAGK, clarified substrate binding and catalysis but shed no light on arginine inhibition of NAGK. We now shed light on arginine inhibition by determining the crystal structures, at 2.75 A and 2.95 A resolution, of arginine-complexed Thermotoga maritima and arginine-free Pseudomonas aeruginosa NAGKs, respectively. Both enzymes are highly similar ring-like hexamers having a central orifice of approximately 30 A diameter. They are formed by linking three E.coli NAGK-like homodimers through the interlacing of an N-terminal mobile kinked alpha-helix, which is absent from E.coli NAGK. Arginine is bound in each subunit of T.maritima NAGK, flanking the interdimeric junction, in a site formed between the N helix and the C lobe of the subunit. This site is also present, in variable conformations, in P.aeruginosa NAGK, but is missing from E.coli NAGK. Arginine, by gluing the C lobe of each subunit to the inter-dimeric junction, may stabilize an enlarged active centre conformation, hampering catalysis. Acetylglutamate counters arginine inhibition by promoting active centre closure. The hexameric architecture justifies the observed sigmoidal arginine inhibition kinetics with a high Hill coefficient (N approximately 4), and appears essential for arginine inhibition and for NAGK-PII complex formation, since this complex may involve binding of NAGK and PII with their 3-fold axes aligned. The NAGK structures allow identification of diagnostic sequence signatures for arginine inhibition. These signatures are found also in the homologous arginine-inhibited enzyme NAG synthase. The findings on NAGK shed light on the structure, function and arginine inhibition of this synthase, for which a hexameric model is constructed.     

Mapping active site residues in glutamate-5-kinase. The substrate glutamate and the feed-back inhibitor proline bind at overlapping sites

Glutamate-5-kinase (G5K) catalyzes the controlling first step of proline biosynthesis. Substrate binding, catalysis and feed-back inhibition by proline are functions of the N-terminal approximately 260-residue domain of G5K. We study here the impact on these functions of 14 site-directed mutations affecting 9 residues of Escherichia coli G5K, chosen on the basis of the structure of the bisubstrate complex of the homologous enzyme acetylglutamate kinase (NAGK). The results support the predicted roles of K10, K217 and T169 in catalysis and ATP binding and of D150 in orienting the catalytic lysines. They support the implication of D148 and D150 in glutamate binding and of D148 and N149 in proline binding. Proline increases the S(0.5) for glutamate and appears to bind at a site overlapping with the site for glutamate. We conclude that G5K and NAGK closely resemble each other concerning substrate binding and catalysis, but that they have different mechanisms of feed-back control.     

Metabolite regulation of the interaction between Arabidopsis thaliana PII and N-acetyl-l-glutamate kinase

The metabolic control of the interaction between ArabidopsisN-acetyl-l-glutamate kinase (NAGK) and the PII protein has been studied. Both gel exclusion and affinity chromatography analyses of recombinant, affinity-purified PII (trimeric complex) and NAGK (hexameric complex) showed that NAGK strongly interacted with PII only in the presence of Mg-ATP, and that this process was reversed by 2-oxoglutarate (2-OG). Furthermore, metabolites such as arginine, glutamate, citrate, and oxalacetate also exerted a negative effect on the PII-NAGK complex formation in the presence of Mg-ATP. Using chloroplast protein extracts and PII affinity chromatography, NAGK interacted with PII only in the presence of ATP-Mg²⁺, and this process was antagonized by 2-OG. These results reveal a complex metabolic control of the PII interaction with NAGK in the chloroplast stroma of higher plants.     

Three-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding

l-Alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to l-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 Å resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16° toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism.     

The Structure of Sedoheptulose-7-Phosphate Isomerase from Burkholderia pseudomallei Reveals a Zinc Binding Site at the Heart of the Active Site

Heptoses are found in the surface polysaccharides of most bacteria, contributing to structures that are essential for virulence and antibiotic resistance. Consequently, the biosynthetic enzymes for these sugars are attractive targets for novel antibiotics. The best characterized biosynthetic enzyme is GmhA, which catalyzes the conversion of sedoheptulose-7-phosphate into d-glycero-d-manno-heptopyranose-7-phosphate, the first step in the biosynthesis of heptose. Here, the structure of GmhA from Burkholderia pseudomallei is reported. This enzyme contains a zinc ion at the heart of its active site: this ion stabilizes the active, closed form of the enzyme and presents coordinating side chains as a potential acid and base to drive catalysis. A complex with the product demonstrates that the enzyme retains activity in the crystal and thus suggests that the closed conformation is catalytically relevant and is an excellent target for the development of therapeutics. A revised mechanism for the action of GmhA is postulated on the basis of this structure and the activity of B. pseudomallei GmhA mutants.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Ring-opening mechanism revealed by crystal structures of NagB and its ES intermediate complex

Glucosamine 6-phosphate deaminase (NagB) catalyzes the conversion of d-glucosamine 6-phosphate (GlcN6P) to d-fructose 6-phosphate and ammonia. This reaction is the final step of N-acetylglucosamine utilization and decides its metabolic fate. The enzyme from Streptococcus mutans belongs to the monomeric subfamily of NagB. The crystal structure of the native SmuNagB (NagB from S. mutans) presented here, compared with the structures of its homologs BsuNagB (NagB from Bacillus subtilis) and EcoNagB (NagB from E. coli), implies a conformational change of the ‘lid’ motif in the activation of the monomeric NagB enzyme. We have also captured the enzyme-substrate intermediate complex of the NagB family at low pH, where a remarkable loss of the catalytic activity of SmuNagB was detected. The enzyme-substrate intermediate presents the initial step of the GlcN6P deaminase reaction. The structural evidence (1) supports the α-anomer of GlcN6P as the specific natural substrate of NagB; (2) displays the substrate-binding pocket at the active site; and (3) together with the site-directed mutagenesis studies, demonstrates the ring-opening mechanism of an Asn-His-Glu triad that performs the proton transfer from O1 to O5 to open the sugar ring.     

The structures of Thermoplasma volcanium phosphoribosyl pyrophosphate synthetase bound to ribose-5-phosphate and ATP analogs

Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P2₁. The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP-Mg²⁺ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively.     

D-ribose-5-phosphate isomerase B from Escherichia coli is also a functional D-allose-6-phosphate isomerase, while the Mycobacterium tuberculosis enzyme is not

Interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate is an important step in the pentose phosphate pathway. Two unrelated enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars d-allose-6-phosphate (All6P) and d-allulose-6-phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme's primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-d-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme's structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate ribose-5-phosphate, shed additional light on the reaction mechanism of RpiBs generally.     

Crystal structure of YihS in complex with D-mannose: structural annotation of Escherichia coli and Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerase

The three-dimensional structure of a Salmonella enterica hypothetical protein YihS is significantly similar to that of N-acyl-d-glucosamine 2-epimerase (AGE) with respect to a common scaffold, an α₆/α₆-barrel, although the function of YihS remains to be clarified. To identify the function of YihS, Escherichia coli and S. enterica YihS proteins were overexpressed in E. coli, purified, and characterized. Both proteins were found to show no AGE activity but showed cofactor-independent aldose-ketose isomerase activity involved in the interconversion of monosaccharides, mannose, fructose, and glucose, or lyxose and xylulose. In order to clarify the structure/function relationship of YihS, we determined the crystal structure of S. enterica YihS mutant (H248A) in complex with a substrate (d-mannose) at 1.6 Å resolution. This enzyme-substrate complex structure is the first demonstration in the AGE structural family, and it enables us to identify active-site residues and postulate a reaction mechanism for YihS. The substrate, β-d-mannose, fits well in the active site and is specifically recognized by the enzyme. The substrate-binding site of YihS for the mannose C1 and O5 atoms is architecturally similar to those of mutarotases, suggesting that YihS adopts the pyranose ring-opening process by His383 and acidifies the C2 position, forming an aldehyde at the C1 position. In the isomerization step, His248 functions as a base catalyst responsible for transferring the proton from the C2 to C1 positions through a cis-enediol intermediate. On the other hand, in AGE, His248 is thought to abstract and re-adduct the proton at the C2 position of the substrate. These findings provide not only molecular insights into the YihS reaction mechanism but also useful information for the molecular design of novel carbohydrate-active enzymes with the common scaffold, α₆/α₆-barrel.     

Gene cloning and catalysis features of a new mannitol-1-phosphate dehydrogenase (BbMPD) from Beauveria bassiana

A long-chain mannitol-1-phosphate dehydrogenase (MPD) was characterized for the first time from fungal entomopathogen Beauveria bassiana by gene cloning, heterogeneous expression and activity analysis. The cloned gene BbMPD consisted of a 1334-bp open reading frame (ORF) with a 158-bp intron and the 935-bp upstream and 780-bp downstream regions. The ORF-encoded 391-aa protein (42 kDa) showed less than 75% sequence identity to 17 fungal MPDs documented and shared two conserved domains with the fungal MPD family at the N- and C-terminus, respectively. The new enzyme was expressed well in the Luria-Bertani culture of engineered Escherichia coli BL21 by 16-h induction of 0.5 mM isopropyl 1-thio-β-d-galactopyranoside at 20 °C after 5-h growth at 37 °C. The purified BbMPD exhibited a high catalytic efficiency (k_cat/K_m) of 1.31 × 10⁴ mM⁻¹ s⁻¹ in the reduction of the highly specific substrate d-fructose-6-phosphate to d-mannitol-1-phosphate. Its activity was maximal at the reaction regime of 37 °C and pH 7.0 and was much more sensitive to Cu²⁺ and Zn²⁺ than to Li⁺ and Mn²⁺. The results indicate a crucial role of BbMPD in the mannitol biosynthesis of B. bassiana.     

First structures of an active bacterial tyrosinase reveal copper plasticity

Tyrosinase is a member of the type 3 copper enzyme family that is involved in the production of melanin in a wide range of organisms. The crystal structures of a tyrosinase from Bacillus megaterium were determined at a resolution of 2.0-2.3 Å. The enzyme crystallized as a dimer in the asymmetric unit and was shown to be active in crystal. The overall monomeric structure is similar to that of the monomer of the previously determined tyrosinase from Streptomyces castaneoglobisporus, but it does not contain an accessory Cu-binding “caddie” protein. Two Cu(II) ions, serving as the major cofactors within the active site, are coordinated by six conserved histidine residues. However, determination of structures under different conditions shows varying occupancies and positions of the copper ions. This apparent mobility in copper binding modes indicates that there is a pathway by which copper is accumulated or lost by the enzyme. Additionally, we suggest that residues R209 and V218, situated in a second shell of residues surrounding the active site, play a role in substrate binding orientation based on their flexibility and position. The determination of a structure with the inhibitor kojic acid, the first tyrosinase structure with a bound ligand, revealed additional residues involved in the positioning of substrates in the active site. Comparison of wild-type structures with the structure of the site-specific variant R209H, which possesses a higher monophenolase/diphenolase activity ratio, lends further support to a previously suggested mechanism by which monophenolic substrates dock mainly to CuA.     

The structures of L-rhamnose isomerase from Pseudomonas stutzeri in complexes with L-rhamnose and D-allose provide insights into broad substrate specificity

Pseudomonas stutzeril-rhamnose isomerase (P. stutzeri L-RhI) can efficiently catalyze the isomerization between various aldoses and ketoses, showing a broad substrate specificity compared to L-RhI from Escherichia coli (E. coli L-RhI). To understand the relationship between structure and substrate specificity, the crystal structures of P. stutzeri L-RhI alone and in complexes with l-rhamnose and d-allose which has different configurations of C4 and C5 from l-rhamnose, were determined at a resolution of 2.0 Å, 1.97 Å, and 1.97 Å, respectively. P. stutzeri L-RhI has a large domain with a (β/α)₈ barrel fold and an additional small domain composed of seven α-helices, forming a homo tetramer, as found in E. coli L-RhI and d-xylose isomerases (D-XIs) from various microorganisms. The β1-α1 loop (Gly60-Arg76) of P. stutzeri L-RhI is involved in the substrate binding of a neighbouring molecule, as found in D-XIs, while in E. coli L-RhI, the corresponding β1-α1 loop is extended (Asp52-Arg78) and covers the substrate-binding site of the same molecule. The complex structures of P. stutzeri L-RhI with l-rhamnose and d-allose show that both substrates are nicely fitted to the substrate -binding site. The part of the substrate-binding site interacting with the substrate at the 1, 2, and 3 positions is equivalent to E. coli L-RhI, and the other part interacting with the 4, 5, and 6 positions is similar to D-XI. In E. coli L-RhI, the β1-α1 loop creates an unique hydrophobic pocket at the the 4, 5, and 6 positions, leading to the strictly recognition of l-rhamnose as the most suitable substrate, while in P. stutzeri L-RhI, there is no corresponding hydrophobic pocket where Phe66 from a neighbouring molecule merely forms hydrophobic interactions with the substrate, leading to the loose substrate recognition at the 4, 5, and 6 positions.     

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a K_m of 41 mM and a V_max of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.     

The C-terminal of CysM from Mycobacterium tuberculosis protects the aminoacrylate intermediate and is involved in sulfur donor selectivity

A new crystal structure of the dimeric cysteine synthase CysM from Mycobacterium tuberculosis reveals an open and a closed conformation of the enzyme. In the closed conformation the five carboxy-terminal amino acid residues are inserted into the active site cleft. Removal of this segment results in a decreased lifetime of the α-aminoacrylate reaction intermediate, an increased sensitivity to oxidants such as hydrogen peroxide, and loss of substrate selectivity with respect to the sulfur carrier thiocarboxylated CysO. These results highlight features of CysM that might be of particular importance for cysteine biosynthesis under oxidative stress in M. tuberculosis.     

Crystal structures of ADP and AMPPNP-bound propionate kinase (TdcD) from Salmonella typhimurium: comparison with members of acetate and sugar kinase/heat shock cognate 70/actin superfamily

Recently, it has been shown that l-threonine can be catabolized non-oxidatively to propionate via 2-ketobutyrate. Propionate kinase (TdcD; EC 2.7.2.-) catalyses the last step of this metabolic process by enabling the conversion of propionyl phosphate and ADP to propionate and ATP. To provide insights into the substrate-binding pocket and catalytic mechanism of TdcD, the crystal structures of the enzyme from Salmonella typhimurium in complex with ADP and AMPPNP have been determined to resolutions of 2.2A and 2.3A, respectively, by molecular replacement using Methanosarcina thermophila acetate kinase (MAK; EC 2.7.2.1). Propionate kinase, like acetate kinase, contains a fold with the topology betabetabetaalphabetaalphabetaalpha, identical with that of glycerol kinase, hexokinase, heat shock cognaten 70 (Hsc70) and actin, the superfamily of phosphotransferases. The structure consists of two domains with the active site contained in a cleft at the domain interface. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. This was further confirmed by kinetic studies with the purified enzyme, which showed about ten times lower K(m) for propionate (2.3 mM) than for acetate (26.9 mM). Comparison of TdcD complex structures with those of acetate and sugar kinase/Hsc70/actin obtained with different ligands has permitted the identification of catalytically essential residues involved in substrate binding and catalysis, and points to both structural and mechanistic similarities. In the well-characterized members of this superfamily, ATP phosphoryl transfer or hydrolysis is coupled to a large conformational change in which the two domains close around the active site cleft. The significant amino acid sequence similarity between TdcD and MAK has facilitated study of domain movement, which indicates that the conformation assumed by the two domains in the nucleotide-bound structure of TdcD may represent an intermediate point in the pathway of domain closure.     

Crystal Structure of Bacillus cereusd-Alanyl Carrier Protein Ligase (DltA) in Complex with ATP

d-Alanylation of lipoteichoic acids modulates the surface charge and ligand binding of the Gram-positive cell wall. Disruption of the bacterial dlt operon involved in teichoic acid alanylation, as well as inhibition of the DltA (d-alanyl carrier protein ligase) protein, has been shown to render the bacterium more susceptible to conventional antibiotics and host defense responses. The DltA catalyzes the adenylation and thiolation reactions of d-alanine. This enzyme belongs to a superfamily of AMP-forming domains such as the ubiquitous acetyl-coenzyme A synthetase. We have determined the 1.9-Å-resolution crystal structure of a DltA protein from Bacillus cereus in complex with ATP. This structure sheds light on the geometry of the bound ATP. The invariant catalytic residue Lys492 appears to be mobile, suggesting a molecular mechanism of catalysis for this superfamily of enzymes. Specific roles are also revealed for two other invariant residues: the divalent cation-stabilizing Glu298 and the β-phosphate-interacting Arg397. Mutant proteins with a glutamine substitution at position 298 or 397 are inactive.     

Half-of-the-sites reactivity of transketolase from Saccharomyces cerevisiae

Cleavage by yeast transketolase of the donor substrate, d-xylulose 5-phosphate, in the absence of the acceptor substrate was studied using stopped-flow spectrophotometry. One mole of the substrate was shown to be cleaved in the prestationary phase, leading to the formation of one mole of the reaction product per mole enzyme, which has two active centers. This observation indicates that only one out of the two active centers functions (i.e., binds and cleaves the substrate) at a time. Such half-of-the-sites reactivity of transketolase conforms well with our understanding, proposed previously, that the active centers of the enzyme operate in sequence (in phase opposition): the cleavage of a ketose within one center (first phase of the transketolase reaction) is paralleled by its formation in the other center (glycolaldehyde residue is condensed with the acceptor substrate, and the second stage of the transketolase reaction is thereby completed) [M.V. Kovina, G.A. Kochetov, FEBS Lett. 440 (1998) 81-84].