Crystal structures of the elusive Rhizobium etli L-asparaginase reveal a peculiar active site.

Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes an essential L-asparaginase (ReAV) with no sequence homology to known enzymes with this activity. High-resolution crystal structures of ReAV show indeed a structurally distinct, dimeric enzyme, with some resemblance to glutaminases and ß-lactamases. However, ReAV has no glutaminase or lactamase activity, and at pH 9 its allosteric asparaginase activity is relatively high, with Km for L-Asn at 4.2 mM and kcat of 438 s-1. The active site of ReAV, deduced from structural comparisons and confirmed by mutagenesis experiments, contains a highly specific Zn2+ binding site without a catalytic role. The extensive active site includes residues with unusual chemical properties. There are two Ser-Lys tandems, all connected through a network of H-bonds to the Zn center, and three tightly bound water molecules near Ser48, which clearly indicate the catalytic nucleophile.

Characterization of intra- and inter-species hybrid tetramers of pyruvate carboxylase: Biotin and the BCCP domain play a crucial role in determination of the kinetics and thermodynamics of catalysis.

The formation, kinetics and thermodynamic activation parameters of hybrid tetramers of pyruvate carboxylase (PC) formed between wild-type Rhizobium etli pyruvate carboxylase (WTRePC) and mutant forms of this enzyme, as well as between Aspergillus nidulans PC and mutant forms of RePC have been characterized in a previous study. In this current work, we aim to extend the previous study by forming hybrid tetramers between WTRePC or chicken liver PC (CLPC) with single or double mutant RePCs. By forming hybrid tetramers between WTRePC with either K1119A or ΔBCCP RePC, the biotin moiety and BCCP (biotin carboxyl carrier protein) domain appear to play a crucial role in determination of thermodynamic activation parameters, especially the activation entropy, and the order of tetrameric structure. Using E218A:K1119A hybrid tetramers, an alternative pathway of biotin carboxylation occurred only in the absence of acetyl CoA. In this pathway, the biotin of the E218A subunits is carboxylated in the BC domain of the K1119A subunits, since the E218A mutation destroys the catalytic activity of the BC domain. Transfer of the carboxyl group to pyruvate could then occur in the CT domain of either E218A or K1119A. Part of the reduction of activity in hybrid tetramers of WTRePC and double mutant, E218A.K1119A could result from the loss of this pathway. Previously, D1018A mutant RePC homotetramers exhibited a 12-fold increase in the rate constant for catalysis in the absence of acetyl CoA. This was taken to indicate that inter-residue interactions involving D1018 inhibit the interconversion between the symmetrical and asymmetrical forms of the tetramer in the absence of acetyl CoA. The mutation, D1018A, in hybrid tetramers of WTRePC:D1018A.K1119A (D1018A.K1119A is a double mutant form of RePC) had no such effect on the rate constant, suggesting that in hybrid tetramers obligatory oscillation between asymmetrical and symmetrical conformers of the tetramer is not required to drive the catalytic cycle. Finally, K1119A or E218A RePC mutant can form hybrid tetramers with PC subunits from an evolutionarily distant species, chicken, that have stability characteristics that lie between those of the homotetramers of the two enzymes. This work provides insights into the how the PC tetramer functions to perform catalysis and is regulated by acetyl CoA. The ability to form hybrid tetrameric PCs composed of PC subunits from widely varying species that have a mixture of characteristics of the two source enzymes may also provide ways of developing novel PCs for biotechnological purposes.

Allosteric regulation alters carrier domain translocation in pyruvate carboxylase.

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. The reaction occurs in two separate catalytic domains, coupled by the long-range translocation of a biotinylated carrier domain (BCCP). Here, we use a series of hybrid PC enzymes to examine multiple BCCP translocation pathways in PC. These studies reveal that the BCCP domain of PC adopts a wide range of translocation pathways during catalysis. Furthermore, the allosteric activator, acetyl CoA, promotes one specific intermolecular carrier domain translocation pathway. These results provide a basis for the ordered thermodynamic state and the enhanced carboxyl group transfer efficiency in the presence of acetyl CoA, and reveal that the allosteric effector regulates enzyme activity by altering carrier domain movement. Given the similarities with enzymes involved in the modular synthesis of natural products, the allosteric regulation of carrier domain movements in PC is likely to be broadly applicable to multiple important enzyme systems.

A high-throughput screening assay for pyruvate carboxylase.

Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate (OAA), an important metabolic reaction in a wide range of organisms. Small molecules directed against PC would enable detailed studies on the metabolic role of this enzyme and would have the potential to be developed into pharmacological agents. Currently, specific and potent small molecule regulators of PC are unavailable. To assist in efforts to find, develop, and characterize small molecule effectors of PC, a novel fixed-time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored adduct with an absorbance maximum at 530 nm. This fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobium etli PC, Staphylococcus aureus PC, and Listeria monocytogenes PC, and is highly amenable to high-throughput screening. The assay was validated using a plate uniformity assessment test and a pilot screen of a library of 1280 compounds. The results indicate that the assay is suitable for screening small molecule libraries to find novel small molecule effectors of PC.

Synthesis of N-acetyl-d-quinovosamine in Rhizobium etli CE3 is completed after its 4-keto-precursor is linked to a carrier lipid.

Bacterial O-antigens are synthesized on lipid carriers before being transferred to lipopolysaccharide core structures. Rhizobium etli CE3 lipopolysaccharide is a model for understanding O-antigen biological function. CE3 O-antigen structure and genetics are known. However, proposed enzymology for CE3 O-antigen synthesis has been examined very little in vitro, and even the sugar added to begin the synthesis is uncertain. A model based on mutagenesis studies predicts that 2-acetamido-2,6-dideoxy-d-glucose (QuiNAc) is the first O-antigen sugar and that genes wreV, wreQ and wreU direct QuiNAc synthesis and O-antigen initiation. Previously, synthesis of UDP-QuiNAc was shown to occur in vitro with a WreV orthologue (4,6-hexose dehydratase) and WreQ (4-reductase), but the WreQ catalysis in this conventional deoxyhexose-synthesis pathway was very slow. This seeming deficiency was explained in the present study after WreU transferase activity was examined in vitro. Results fit the prediction that WreU transfers sugar-1-phosphate to bactoprenyl phosphate (BpP) to initiate O-antigen synthesis. Interestingly, WreU demonstrated much higher activity using the product of the WreV catalysis [UDP-4-keto-6-deoxy-GlcNAc (UDP-KdgNAc)] as the sugar-phosphate donor than using UDP-QuiNAc. Furthermore, the WreQ catalysis with WreU-generated BpPP-KdgNAc as the substrate was orders of magnitude faster than with UDP-KdgNAc. The inferred product BpPP-QuiNAc reacted as an acceptor substrate in an in vitro assay for addition of the second O-antigen sugar, mannose. These results imply a novel pathway for 6-deoxyhexose synthesis that may be commonly utilized by bacteria when QuiNAc is the first sugar of a polysaccharide or oligosaccharide repeat unit: UDP-GlcNAc → UDP-KdgNAc → BpPP-KdgNAc → BpPP-QuiNAc.

Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli.

BACKGROUND: The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system. RESULTS: To fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a "landing pad" (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %. CONCLUSIONS: Integration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.

Investigation of the Roles of Allosteric Domain Arginine, Aspartate, and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in Relation to Its Activation by Acetyl CoA.

The mechanism of allosteric activation of pyruvate carboxylase by acetyl CoA is not fully understood. Here we have examined the roles of residues near the acetyl CoA binding site in the allosteric activation of Rhizobium etli pyruvate carboxylase using site-directed mutagenesis. Arg429 was found to be especially important for acetyl CoA binding as substitution with serine resulted in a 100-fold increase in the Ka of acetyl CoA activation and a large decrease in the cooperativity of this activation. Asp420 and Arg424, which do not make direct contact with bound acetyl CoA, were nonetheless found to affect acetyl CoA binding when mutated, probably through changed interactions with another acetyl CoA binding residue, Arg427. Thermodynamic activation parameters for the pyruvate carboxylation reaction were determined from modified Arrhenius plots and showed that acetyl CoA acts to decrease the activation free energy of the reaction by both increasing the activation entropy and decreasing the activation enthalpy. Most importantly, mutations of Asp420, Arg424, and Arg429 enhanced the activity of the enzyme in the absence of acetyl CoA. A main focus of this work was the detailed investigation of how this increase in activity occurred in the R424S mutant. This mutation decreased the activation enthalpy of the pyruvate carboxylation reaction by an amount consistent with removal of a single hydrogen bond. It is postulated that Arg424 forms a hydrogen bonding interaction with another residue that stabilizes the asymmetrical conformation of the R. etli pyruvate carboxylase tetramer, constraining its interconversion to the symmetrical conformer that is required for catalysis.

Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation.

We have examined the roles of Asp1018, Glu1027, Arg469 and Asp471 in the allosteric domain of Rhizobium etli pyruvate carboxylase. Arg469 and Asp471 interact directly with the allosteric activator acetyl coenzyme A (acetyl CoA) and the R469S and R469K mutants showed increased enzymic activity in the presence and absence of acetyl CoA, whilst the D471A mutant exhibited no acetyl CoA-activation. E1027A, E1027R and D1018A mutants had increased activity in the absence of acetyl CoA, but not in its presence. These results suggest that most of these residues impose restrictions on the structure and/or dynamics of the enzyme to affect activity.

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.

Mechanisms of inhibition of Rhizobium etli pyruvate carboxylase by L-aspartate.

L-aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of L-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase. Kinetic studies demonstrated that L-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. L-aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of L-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of L-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxamate-induced oxaloacetate decarboxylation reaction that occurs in the carboxyl transferase domain. L-aspartate was shown to competitively inhibit bicarbonate-dependent MgATP cleavage with respect to MgATP but also probably inhibits carboxybiotin formation and/or translocation of the carboxybiotin to the site of pyruvate carboxylation. Unlike acetyl-CoA, L-aspartate has no effect on the coupling between MgATP cleavage and oxaloacetate formation. The results suggest that the three allosteric effector sites (acetyl-CoA, MgTNP-ATP, and L-aspartate) are spatially distinct but connected by a network of allosteric interactions.

The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase.

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes.

In vitro biosynthesis and chemical identification of UDP-N-acetyl-d-quinovosamine (UDP-d-QuiNAc).

N-acetyl-d-quinovosamine (2-acetamido-2,6-dideoxy-d-glucose, QuiNAc) occurs in the polysaccharide structures of many Gram-negative bacteria. In the biosynthesis of QuiNAc-containing polysaccharides, UDP-QuiNAc is the hypothetical donor of the QuiNAc residue. Biosynthesis of UDP-QuiNAc has been proposed to occur by 4,6-dehydration of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) to UDP-2-acetamido-2,6-dideoxy-d-xylo-4-hexulose followed by reduction of this 4-keto intermediate to UDP-QuiNAc. Several specific dehydratases are known to catalyze the first proposed step. A specific reductase for the last step has not been demonstrated in vitro, but previous mutant analysis suggested that Rhizobium etli gene wreQ might encode this reductase. Therefore, this gene was cloned and expressed in Escherichia coli, and the resulting His6-tagged WreQ protein was purified. It was tested for 4-reductase activity by adding it and NAD(P)H to reaction mixtures in which 4,6-dehydratase WbpM had acted on the precursor substrate UDP-GlcNAc. Thin layer chromatography of the nucleotide sugars in the mixture at various stages of the reaction showed that WbpM converted UDP-GlcNAc completely to what was shown to be its 4-keto-6-deoxy derivative by NMR and that addition of WreQ and NADH led to formation of a third compound. Combined gas chromatography-mass spectrometry analysis of acid hydrolysates of the final reaction mixture showed that a quinovosamine moiety had been synthesized after WreQ addition. The two-step reaction progress also was monitored in real time by NMR. The final UDP-sugar product after WreQ addition was purified and determined to be UDP-d-QuiNAc by one-dimensional and two-dimensional NMR experiments. These results confirmed that WreQ has UDP-2-acetamido-2,6-dideoxy-d-xylo-4-hexulose 4-reductase activity, completing a pathway for UDP-d-QuiNAc synthesis in vitro.

Coordinating role of His216 in MgATP binding and cleavage in pyruvate carboxylase.

His216 is a well-conserved residue in pyruvate carboxylases and, on the basis of structures of the enzyme, appears to have a role in the binding of MgATP, forming an interaction with the 3'-hydroxyl group of the ribose ring. Mutation of this residue to asparagine results in a 9-fold increase in the Km for MgATP in its steady-state cleavage in the absence of pyruvate and a 3-fold increase in the Km for MgADP in its steady-state phosphorylation by carbamoyl phosphate. However, from single-turnover experiments of MgATP cleavage, the Kd of the enzyme·MgATP complex is essentially the same in the wild-type enzyme and H216N. Direct stopped-flow measurements of nucleotide binding and release using the fluorescent analogue FTP support these observations. However, the first-order rate constant for MgATP cleavage in the single-turnover experiments in H216N is only 0.75% of that for the wild-type enzyme, and thus, the MgATP cleavage step is rate-limiting in the steady state for H216N but not for the wild-type enzyme. Close examination of the structure of the enzyme suggested that His216 may also interact with Glu218, which in turn interacts with Glu305 to form a proton relay system involved in the deprotonation of bicarbonate. Single-turnover MgATP cleavage experiments with mutations of these two residues resulted in kinetic parameters similar to those observed in H216N. We suggest that the primary role of His216 is to coordinate the binding of MgATP and the deprotonation of bicarbonate in the reaction to form the putative carboxyphosphate intermediate by participation in a proton relay system involving Glu218 and Glu305.

Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs.

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates.

The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation.

The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a beta-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection.

Characterization of IntA, a bidirectional site-specific recombinase required for conjugative transfer of the symbiotic plasmid of Rhizobium etli CFN42.

Site-specific recombination occurs at short specific sequences, mediated by the cognate recombinases. IntA is a recombinase from Rhizobium etli CFN42 and belongs to the tyrosine recombinase family. It allows cointegration of plasmid p42a and the symbiotic plasmid via site-specific recombination between attachment regions (attA and attD) located in each replicon. Cointegration is needed for conjugative transfer of the symbiotic plasmid. To characterize this system, two plasmids harboring the corresponding attachment sites and intA were constructed. Introduction of these plasmids into R. etli revealed IntA-dependent recombination events occurring at high frequency. Interestingly, IntA promotes not only integration, but also excision events, albeit at a lower frequency. Thus, R. etli IntA appears to be a bidirectional recombinase. IntA was purified and used to set up electrophoretic mobility shift assays with linear fragments containing attA and attD. IntA-dependent retarded complexes were observed only with fragments containing either attA or attD. Specific retarded complexes, as well as normal in vivo recombination abilities, were seen even in derivatives harboring only a minimal attachment region (comprising the 5-bp central region flanked by 9- to 11-bp inverted repeats). DNase I-footprinting assays with IntA revealed specific protection of these zones. Mutations that disrupt the integrity of the 9- to 11-bp inverted repeats abolish both specific binding and recombination ability, while mutations in the 5-bp central region severely reduce both binding and recombination. These results show that IntA is a bidirectional recombinase that binds to att regions without requiring neighboring sequences as enhancers of recombination.

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 Asp(590) and Tyr(628) 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.

Oxamate is an alternative substrate for pyruvate carboxylase from Rhizobium etli.

Oxamate, an isosteric and isoelectronic inhibitory analogue of pyruvate, enhances the rate of enzymatic decarboxylation of oxaloacetate in the carboxyl transferase domain of pyruvate carboxylase (PC). It is unclear, though, how oxamate exerts a stimulatory effect on the enzymatic reaction. Herein, we report direct (13)C nuclear magnetic resonance (NMR) evidence that oxamate acts as a carboxyl acceptor, forming a carbamylated oxamate product and thereby accelerating the enzymatic decarboxylation reaction. (13)C NMR was used to monitor the PC-catalyzed formation of [4-(13)C]oxaloacetate and subsequent transfer of (13)CO(2) from oxaloacetate to oxamate. In the presence of oxamate, the apparent K(m) for oxaloacetate is artificially suppressed (from 15 to 4-5 µM). Interestingly, the steady-state kinetic analysis of the initial rates determined at varying concentrations of oxaloacetate and fixed concentrations of oxamate revealed initial velocity patterns inconsistent with a simple ping-pong-type mechanism. Rather, the patterns suggest the existence of an alternate decarboxylation pathway in which an unstable intermediate is formed. The steady-state kinetic analysis coupled with the normal (13)(V/K) kinetic isotope effect observed on C-4 of oxaloacetate [(13)(V/K) = 1.0117 ± 0.0005] indicates that the transfer of CO(2) from carboxybiotin to oxamate is the partially rate-limiting step of the enzymatic reaction. The catalytic mechanism proposed for the carboxylation of oxamate is similar to that proposed for the carboxylation of pyruvate, which occurs via the formation of an enol intermediate.

Roles of predicted glycosyltransferases in the biosynthesis of the Rhizobium etli CE3 O antigen.

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer. The genetic regions required for its synthesis have been identified, and the nucleotide sequences are known. The structure of the O antigen has been determined, but the roles of specific genes in synthesizing this structure are relatively unclear. Within the known O-antigen genetic clusters of this strain, nine open reading frames (ORFs) were found to contain a conserved glycosyltransferase domain. Each ORF was mutated, and the resulting mutant lipopolysaccharide (LPS) was analyzed. Tricine SDS-PAGE revealed stepwise truncations of the O antigen that were consistent with differences in mutant LPS sugar compositions and reactivity with O-antigen-specific monoclonal antibodies. Based on these results and current theories of O-antigen synthesis, specific roles were deduced for each of the nine glycosyltransferases, and a model for biosynthesis of the R. etli CE3 O antigen was proposed. In this model, O-antigen biosynthesis is initiated with the addition of N-acetyl-quinovosamine-phosphate (QuiNAc-P) to bactoprenol-phosphate by glycosyltransferase WreU. Glycosyltransferases WreG, WreE, WreS, and WreT would each act once to attach mannose, fucose, a second fucose, and 3-O-methyl-6-deoxytalose (3OMe6dTal), respectively. WreH would then catalyze the addition of methyl glucuronate (MeGlcA) to complete the first instance of the O-antigen repeat unit. Four subsequent repeats of this unit composed of fucose, 3OMe6dTal, and MeGlcA would be assembled by a cycle of reactions catalyzed by two additional glycosyltransferases, WreM and WreL, along with WreH. Finally, the O antigen would be capped by attachment of di- or tri-O-methylated fucose as catalyzed by glycosyltransferase WreB.

Rhizobium etli asparaginase II: an alternative for acute lymphoblastic leukemia (ALL) treatment.

Bacterial L-asparaginase has been a universal component of therapies for childhood acute lymphoblastic leukemia since the 1970s. Two principal enzymes derived from Escherichia coli and Erwinia chrysanthemi are the only options clinically approved to date. We recently reported a study of recombinant L-asparaginase (AnsA) from Rhizobium etli and described an increasing type of AnsA family members. Sequence analysis revealed four conserved motifs with notable differences with respect to the conserved regions of amino acid sequences of type I and type II L-asparaginases, particularly in comparison with therapeutic enzymes from E. coli and E. chrysanthemi. These differences suggested a distinct immunological specificity. Here, we report an in silico analysis that revealed immunogenic determinants of AnsA. Also, we used an extensive approach to compare the crystal structures of E. coli and E. chrysantemi asparaginases with a computational model of AnsA and identified immunogenic epitopes. A three-dimensional model of AsnA revealed, as expected based on sequence dissimilarities, completely different folding and different immunogenic epitopes. This approach could be very useful in transcending the problem of immunogenicity in two major ways: by chemical modifications of epitopes to reduce drug immunogenicity, and by site-directed mutagenesis of amino acid residues to diminish immunogenicity without reduction of enzymatic activity.