Methylcitric Acid Cycle Research Articles

In-silico characterization and structure-based functional annotation of a hypothetical protein from Campylobacter jejuni involved in propionate catabolism.

Campylobacter jejuni is one of the most prevalent organisms associated with foodborne illness across the globe causing campylobacteriosis and gastritis. Many proteins of C. jejuni are still unidentified. The purpose of this study was to determine the structure and function of a non-annotated hypothetical protein (HP) from C. jejuni. A number of properties like physiochemical characteristics, 3D structure, and functional annotation of the HP (accession No. CAG2129885.1) were predicted using various bioinformatics tools followed by further validation and quality assessment. Moreover, the protein-protein interactions and active site were obtained from the STRING and CASTp server, respectively. The hypothesized protein possesses various characteristics including an acidic pH, thermal stability, water solubility, and cytoplasmic distribution. While alpha-helix and random coil structures are the most prominent structural components of this protein, most of it is formed of helices and coils. Along with expected quality, the 3D model has been found to be novel. This study has identified the potential role of the HP in 2-methylcitric acid cycle and propionate catabolism. Furthermore, protein-protein interactions revealed several significant functional partners. The in-silico characterization of this protein will assist to understand its molecular mechanism of action better. The methodology of this study would also serve as the basis for additional research into proteomic and genomic data for functional potential identification.

The PrpF protein of Shewanella oneidensis MR-1 catalyzes the isomerization of 2-methyl-cis-aconitate during the catabolism of propionate via the AcnD-dependent 2-methylcitric acid cycle.

The 2-methylcitric acid cycle (2-MCC) is a common route of propionate catabolism in microorganisms. In Salmonella enterica, the prpBCDE operon encodes most of the 2-MCC enzymes. In other organisms, e.g., Shewanella oneidensis MR-1, two genes, acnD and prpF replace prpD, which encodes 2-methylcitrate dehydratase. We showed that together, S. oneidensis AcnD and PrpF (SoAcnD, SoPrpF) compensated for the absence of PrpD in a S. enterica prpD strain. We also showed that SoAcnD had 2-methylcitrate dehydratase activity and that PrpF has aconitate isomerase activity. Here we report in vitro evidence that the product of the SoAcnD reaction is an isomer of 2-methyl-cis-aconitate (2-MCA], the product of the SePrpD reaction. We show that the SoPrpF protein isomerizes the product of the AcnD reaction into the PrpD product (2-MCA], a known substrate of the housekeeping aconitase (AcnB]. Given that SoPrpF is an isomerase, that SoAcnD is a dehydratase, and the results from in vivo and in vitro experiments reported here, it is likely that 4-methylaconitate is the product of the AcnD enzyme. Results from in vivo studies using a S. enterica prpD strain show that SoPrpF variants with substitutions of residues K73 or C107 failed to support growth with propionate as the sole source of carbon and energy. High-resolution (1.22 Å) three-dimensional crystal structures of PrpFK73E in complex with trans-aconitate or malonate provide insights into the mechanism of catalysis of the wild-type protein.

First Biochemical Characterization of a Methylcitric Acid Cycle from Bacillus subtilis Strain 168.

The genome of Bacillus subtilis strain 168 contains the mother cell metabolic gene (mmg) operon that encodes homologues from the methylcitric acid cycle. We showed that the three genes, mmgDE and yqiQ(mmgF), provide three of the five steps of the methylcitric acid cycle. We also showed that the fourth step can be supplied by citB (aconitase), and we suggest that the fifth missing step, the propionyl-CoA synthetase, is probably skipped because the β-oxidation of methyl-branched fatty acids by the enzymes encoded by mmgABC should produce propionyl-CoA. We also noted interesting enzymology for MmgD and MmgE. First, MmgD is a bifunctional citrate synthase/2-methylcitrate synthase with 2.3-fold higher activity as a 2-methylcitrate synthase. This enzyme catalyzes the formation of either (2S,3R)- or (2R,3S)-2-methylcitrate, but reports of 2-methylcitrate synthases from other species indicated that they produced the (2S,3S) isomer. However, we showed that MmgD and PrpC (from Escherichia coli) in fact produce the same stereoisomer. Second, the MmgE enzyme is not a stereospecific 2-methylcitrate dehydratase because it can dehydrate at least two of the four diastereomers of 2-methylcitrate to yield either (E)-2-methylaconitate or (Z)-2-methylaconitate. We also showed for the first time that the E. coli homologue PrpD exhibited the same lack of stereospecificity. However, the physiological pathways proceed via (Z)-2-methylaconitate, which served as the substrate for the citB enzyme in the synthesis of 2-methylisocitrate. We completed our characterization of this pathway by showing that the 2-methylisocitrate produced by CitB is converted to pyruvate and succinate by the enzyme YqiQ(MmgF).

Proteomic analysis of organic sulfur compound utilisation in Advenella mimigardefordensis strain DPN7T.

2-Mercaptosuccinate (MS) and 3,3´-ditiodipropionate (DTDP) were discussed as precursor substance for production of polythioesters (PTE). Therefore, degradation of MS and DTDP was investigated in Advenella mimigardefordensis strain DPN7T, applying differential proteomic analysis, gene deletion and enzyme assays. Protein extracts of cells cultivated with MS, DTDP or 3-sulfinopropionic acid (SP) were compared with those cultivated with propionate (P) and/or succinate (S). The chaperone DnaK (ratio DTDP/P 9.2, 3SP/P 4.0, MS/S 6.1, DTDP/S 6.2) and a Do-like serine protease (DegP) were increased during utilization of all organic sulfur compounds. Furthermore, a putative bacterioferritin (locus tag MIM_c12960) showed high abundance (ratio DTDP/P 5.3, 3SP/P 3.2, MS/S 4.8, DTDP/S 3.9) and is probably involved in a thiol-specific stress response. The deletion of two genes encoding transcriptional regulators (LysR (MIM_c31370) and Xre (MIM_c31360)) in the close proximity of the relevant genes of DTDP catabolism (acdA, mdo and the genes encoding the enzymes of the methylcitric acid cycle; prpC,acnD, prpF and prpB) showed that these two regulators are essential for growth of A. mimigardefordensis strain DPN7T with DTDP and that they most probably regulate transcription of genes mandatory for this catabolic pathway. Furthermore, proteome analysis revealed a high abundance (ratio MS/S 10.9) of a hypothetical cupin-2-domain containing protein (MIM_c37420). This protein shows an amino acid sequence similarity of 60% to a newly identified MS dioxygenase from Variovorax paradoxus strain B4. Deletion of the gene and the adjacently located transcriptional regulator LysR, as well as heterologous expression of MIM_c37420, the putative mercaptosuccinate dioxygenase (Msdo) from A. mimigardefordensis, showed that this protein is the key enzyme of MS degradation in A. mimigardefordensis strain DPN7T (KM 0.2 mM, specific activity 17.1 μmol mg-1 min-1) and is controlled by LysR (MIM_c37410).

Engineering of Ralstonia eutropha for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with Ralstonia eutropha relies on the addition of propionate during fermentation, and propionate consumption is one of the major factors affecting the cost of PHBV production. In this study, 7 strains were obtained by genetic manipulating the methylcitric acid cycle and the methylmalonyl-CoA pathway in R. eutropha. Disruption of prpC1 and prpC2 genes did not affect cell growth and PHBV accumulation. All 7 strains were able to accumulation high amounts of PHBVs with 3HV fractions of 0.41–29.1mol% during cultivation in flasks. Fermentation in 7.5-L fermenter showed that genetically engineered Rem-8 was able to yield biomass of 132.8 CDWg/L, of which 68.6% were PHBV with 3HV fraction of 26.0mol% in the biopolymer, indicating promising potentials of commercialization in the future.

Comparative transcriptomics in threeMethylophilaceaespecies uncover different strategies for environmental adaptation

We carried out whole transcriptome analysis of three species of Methylophilaceae, Methylotenera mobilis, Methylotenera versatilis and Methylovorus glucosotrophus, in order to determine which metabolic pathways are actively transcribed in cultures grown in laboratory on C1 substrates and how metabolism changes under semi-in situ conditions. Comparative analyses of the transcriptomes were used to probe the metabolic strategies utilized by each of the organisms in the environment. Our analysis of transcript abundance data focused on changes in expression of methylotrophy metabolic modules, as well as on identifying any functional modules with pronounced response to in situ conditions compared to a limited set of laboratory conditions, highlighting their potential role in environmental adaptation. We demonstrate that transcriptional responses to environmental conditions involved both methylotrophy and non-methylotrophy metabolic modules as well as modules responsible for functions not directly connected to central metabolism. Our results further highlight the importance of XoxF enzymes that were previously demonstrated to be highly expressed in situ and proposed to be involved in metabolism of methanol by Methylophilaceae. At the same time, it appears that different species employ different homologous Xox systems as major metabolic modules. This study also reinforces prior observations of the apparent importance of the methylcitric acid cycle in the Methylotenera species and its role in environmental adaptation. High transcription from the respective gene clusters and pronounced response to in situ conditions, along with the reverse expression pattern for the ribulose monophosphate pathway that is the major pathway for carbon assimilation in laboratory conditions suggest that a switch in central metabolism of Methylotenera takes place in response to in situ conditions. The nature of the metabolite(s) processed via this pathway still remains unknown. Of the functions not related to central metabolism, flagellum and fimbria synthesis functions appeared to be of significance for environmental adaptation, based on their high abundance and differential expression. Our data demonstrate that, besides shared strategies, the organisms employed in this study also utilize strategies unique to each species, suggesting that the genomic divergence plays a role in environmental adaptation.

Gene Identification and Functional Analysis of Methylcitrate Synthase in Citric Acid-ProducingAspergillus nigerWU-2223L

Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4Å resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes.

Preliminary X-ray crystallographic analysis of 2-methylcitrate synthase from Salmonella typhimurium.

Analysis of the genomic sequences of Escherichia coli and Salmonella typhimurium has revealed the presence of several homologues of the well studied citrate synthase (CS). One of these homologues has been shown to code for 2-methylcitrate synthase (2-MCS) activity. 2-MCS catalyzes one of the steps in the 2-methylcitric acid cycle found in these organisms for the degradation of propionate to pyruvate and succinate. In the present work, the gene coding for 2-MCS from S. typhimurium (StPrpC) was cloned in pRSET-C vector and overexpressed in E. coli. The protein was purified to homogeneity using Ni-NTA affinity chromatography. The purified protein was crystallized using the microbatch-under-oil method. The StPrpC crystals diffracted X-rays to 2.4 A resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 92.068, b = 118.159, c = 120.659 A, alpha = 60.84, beta = 67.77, gamma = 81.92 degrees . Computation of rotation functions using the X-ray diffraction data shows that the protein is likely to be a decamer of identical subunits, unlike CSs, which are dimers or hexamers.

In Salmonella enterica , 2-Methylcitrate Blocks Gluconeogenesis

Strains of Salmonella enterica serovar Typhimurium LT2 lacking a functional 2-methylcitric acid cycle (2-MCC) display increased sensitivity to propionate. Previous work from our group indicated that this sensitivity to propionate is in part due to the production of 2-methylcitrate (2-MC) by the Krebs cycle enzyme citrate synthase (GltA). Here we report in vivo and in vitro data which show that a target of the 2-MC isomer produced by GltA (2-MC(GltA)) is fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis. Lack of growth due to inhibition of FBPase by 2-MC(GltA) was overcome by increasing the level of FBPase or by micromolar amounts of glucose in the medium. We isolated an fbp allele encoding a single amino acid substitution in FBPase (S123F), which allowed a strain lacking a functional 2-MCC to grow in the presence of propionate. We show that the 2-MC(GltA) and the 2-MC isomer synthesized by the 2-MC synthase (PrpC; 2-MC(PrpC)) are not equally toxic to the cell, with 2-MC(GltA) being significantly more toxic than 2-MC(PrpC). This difference in 2-MC toxicity is likely due to the fact that as a si-citrate synthase, GltA may produce multiple isomers of 2-MC, which we propose are not substrates for the 2-MC dehydratase (PrpD) enzyme, accumulate inside the cell, and have deleterious effects on FBPase activity. Our findings may help explain human inborn errors in propionate metabolism.

Functioning in situ: gene expression in Methylotenera mobilis in its native environment as assessed through transcriptomics

Methylotrophs, organisms able to gain energy and carbon from compounds containing no carbon-carbon bonds, such as methane, methanol and methylated amines, are widespread in nature. However, knowledge of their nutrient preference and their metabolism is mostly based on experiments with cultures grown in defined laboratory conditions. Here, we use transcriptomics to explore the activity of one methylotroph, Methyotenera mobilis in its natural environment, lake sediment from which it has been previously isolated. Cells encapsulated in incubation cassettes were exposed to sediment conditions, with or without supplementation with a carbon/energy source (methylamine), and gene-expression patterns were compared for those cells to patterns for cells incubated in a defined medium supplemented with methylamine. A few specific trends in gene expression were observed at in situ conditions that may be of environmental significance, as follows. Expression of genes for the linear formaldehyde oxidation pathway linked to tetrahydromethanopterin increased, suggesting an important role for this pathway in situ, in contrast to laboratory condition culture, in which the cyclic ribulose monophosphate pathway seemed to be the major route for formaldehyde oxidation. Along with the ribulose monophosphate cycle that is also a major pathway for assimilating C(1) units, the methylcitric acid cycle seemd to be important in situ, suggesting that multicarbon compounds may be the natural carbon and/or energy substrates for M. mobilis, challenging the notion of an obligately methylotrophic lifestyle for this bacterium. We also detected a major switch in expression of genes responsible for the mode of motility between different conditions: from flagellum-enabled motility in defined medium to in situ expression of pili known to be involved in twitching motility and adherence. Overall, this study offers a novel approach for gaining insights into the lifestyle of individual microbes in their native environments.

Disruption of the 2-methylcitric acid cycle and evaluation of poly-3-hydroxybutyrate-co-3-hydroxyvalerate biosynthesis suggest alternate catabolic pathways of propionate inBurkholderia sacchari

The objective of the present work was to evaluate the relevance of the 2-methylcitric acid cycle (2MCC) to the catabolism of propionate in Burkholderia sacchari. Two B. sacchari mutants unable to grow on propionate were obtained: one disrupted in acnM, and the other in acnM and prpC deleted. An operative 2MCC significantly reduces the bacterial ability to incorporate 3-hydroxyvalerate (3HV) into a biodegradable copolyester accumulated from carbohydrates plus propionate. The efficiency of the mutants in converting propionate to 3HV units (Y(3HV/prp)) increased from 0.09 g*g(-1) to 0.81-0.96 g*g(-1), indicating that acnM and prpC are both essential for growth on propionate. None of the mutations resulted in achievement of the maximum theoretical Y3HV/prp (1.35 g*g(-1)). When increasing concentrations of propionate were supplied, decreasing values of Y3HV/prp were observed. The results obtained corroborate the hypothesis of the presence of other propionate catabolic pathways in B. sacchari. The 2MCC would be the more operative pathway, but a second pathway, which remains to be elucidated, would assume more importance under propionate concentrations of 1 g*L(-1) or higher. The efficiency in converting propionate to 3HV units can be improved by decreasing the propionate concentrations, owing to the role of the 2MCC.

Novel pathway for catabolism of the organic sulfur compound 3,3'-dithiodipropionic acid via 3-mercaptopropionic acid and 3-Sulfinopropionic acid to propionyl-coenzyme A by the aerobic bacterium Tetrathiobacter mimigardefordensis strain DPN7.

The hitherto unstudied microbial degradation of the organic disulfide 3,3'-dithiodipropionic acid (DTDP) was investigated with the recently described bacterium Tetrathiobacter mimigardefordensis strain DPN7(T) (DSM 17166(T); LMG 22922(T)), which is able to use DTDP as the sole carbon source for growth. 3-Mercaptopropionic acid (3MP) and 3-sulfinopropionic acid (3SP) were detected in the growth medium and occurred as intermediates during DTDP degradation. To identify genes coding for enzymes of DTDP catabolism, Tn5::mob-induced mutants of T. mimigardefordensis were generated. Screening of transposon mutant libraries yielded many mutants fully or partially impaired in utilizing DTDP as a carbon source. Mapping of the insertion loci in some mutants identified four disrupted open reading frames (ORFs) with putative metabolic functions. The ORFs were assigned function on the basis of homologies with lpdA (EC 1.8.1.4), cdo (EC 1.13.11.20), sucCD (EC 6.2.1.5), and acnB (EC 4.2.1.3). Tn5::mob insertions occurred additionally in the vicinity of heat shock protein-encoding genes. The predicted function of the LpdA homologue in T. mimigardefordensis is cleavage of the disulfide bond of DTDP to form two molecules of 3MP. Cdo catalyzes the conversion of the sulfhydryl group of 3MP, yielding the corresponding sulfinic acid, 3SP. SucCD exhibits thiokinase activity, ligating coenzyme A (CoA) with 3SP to form 3SP-CoA. Afterwards, an elimination of sulfite via a putative desulfinase is expected. acnB encodes a putative 2-methylisocitrate dehydratase. Therefore, a new pathway is proposed for the catabolism of DTDP via 3MP, 3SP, and 3SP-CoA toward propionyl-CoA, which is then further catabolized via the 2-methylcitric acid cycle in T. mimigardefordensis.

Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

We describe the construction and use of two sets of vectors for the over-expression and purification of protein from Escherichia coli. The set of pTEV plasmids (pTEV3, 4, 5) directs the synthesis of a recombinant protein with a N-terminal hexahistidine (His 6) tag that is removable by the tobacco etch virus (TEV) protease. The set of pKLD plasmids (pKLD66, 116) directs the synthesis of a recombinant protein that contains a N-terminal His 6 and maltose-binding protein tag in tandem, which can also be removed with TEV protease. The usefulness of these plasmids is illustrated by the rapid, high-yield purification of the 2-methylcitrate dehydratase (PrpD) protein of Salmonella enterica, and the 2-methylaconitate isomerase (PrpF) protein of Shewanella oneidensis, two enzymes involved in the catabolism of propionate to pyruvate via the 2-methylcitric acid cycle.

The three‐dimensional crystal structure of the PrpF protein of Shewanella oneidensis complexed with trans‐aconitate: Insights into its biological function

In bacteria, the dehydration of 2-methylcitrate to yield 2-methylaconitate in the 2-methylcitric acid cycle is catalyzed by a cofactor-less (PrpD) enzyme or by an aconitase-like (AcnD) enzyme. Bacteria that use AcnD also require the function of the PrpF protein, whose function was previously unknown. To gain insights into the function of PrpF, the three-dimensional crystal structure of the PrpF protein from the bacterium Shewanella oneidensis was solved at 2.0 A resolution. The protein fold of PrpF is strikingly similar to those of the non-PLP-dependent diaminopimelate epimerase from Haemophilus influenzae, a putative proline racemase from Brucella melitensis, and to a recently deposited structure of a hypothetical protein from Pseudomonas aeruginosa. Results from in vitro studies show that PrpF isomerizes trans-aconitate to cis-aconitate. It is proposed that PrpF catalysis of the cis-trans isomerization proceeds through a base-catalyzed proton abstraction coupled with a rotation about C2-C3 bond of 2-methylaconitate, and that residue Lys73 is critical for PrpF function. The newly identified function of PrpF as a non-PLP-dependent isomerase, together with the fact that PrpD-containing bacteria do not require PrpF, suggest that the isomer of 2-methylaconitate that serves as a substrate of aconitase must have the same stereochemistry as that synthesized by PrpD. From this, it follows that the 2-methylaconitate isomer generated by AcnD is not a substrate of aconitase, and that PrpF is required to generate the correct isomer. As a consequence, the isomerase activity of PrpF may now be viewed as an integral part of the 2-methylcitric acid cycle.

2-Methylcitrate-dependent activation of the propionate catabolic operon (prpBCDE) of Salmonella enterica by the PrpR protein

The function of the PrpR protein of Salmonella enterica serovar Typhimurium LT2 was studied in vitro and in vivo. The PrpR protein is a sensor of 2-methylcitrate (2-MC), an intermediate of the 2-methylcitric acid cycle used by this bacterium to convert propionate to pyruvate. PrpR was unresponsive to citrate (a close structural analogue of 2-MC) and to propionate, suggesting that 2-MC, not propionate, is the metabolite that signals the presence of propionate in the environment to S. enterica. prpR alleles encoding mutant proteins with various levels of 2-MC-independent activity were isolated. All lesions causing constitutive PrpR activity were mapped to the N-terminal domain of the protein. Removal of the entire sensing domain resulted in a protein (PrpR(c)) with the highest 2-MC-independent activity. Residue A162 is critical to 2-MC sensing, since the mutant PrpR protein PrpR(A162T) was as active as the PrpR(c) protein in the absence of 2-MC. DNA footprinting studies identified the site in the region between prpR and the prpBCDE operon to which the PrpR protein binds. Analysis of the binding-site sequence revealed two sites with dyad symmetry. Results from DNase I footprinting assays suggested that the PrpR protein may have higher affinity for the site proximal to the P(prpBCDE) promoter.

Role of alpha-methylacyl coenzyme A racemase in the degradation of methyl-branched alkanes by Mycobacterium sp. strain P101.

A new isolate, Mycobacterium sp. strain P101, is capable of growth on methyl-branched alkanes (pristane, phytane, and squalane). Among ca. 10,000 Tn5-derived mutants, we characterized 2 mutants defective in growth on pristane or n-hexadecane. A single copy of Tn5 was found to be inserted into the coding region of mcr (alpha-methylacyl coenzyme A [alpha-methylacyl-CoA] racemase gene) in mutant P1 and into the coding region of mls (malate synthase gene) in mutant H1. Mutant P1 could not grow on methyl-branched alkanes. The recombinant Mcr produced in Escherichia coli was confirmed to catalyze racemization of (R)-2-methylpentadecanoyl-CoA, with a specific activity of 0.21 micromol . min(-1) . mg of protein(-1). Real-time quantitative reverse transcriptase PCR analyses indicated that mcr gene expression was enhanced by the methyl-branched alkanes pristane and squalane. Mutant P1 used (S)-2-methylbutyric acid for growth but did not use the racemic compound, and growth on n-hexadecane was not inhibited by pristane. These results suggested that the oxidation of the methyl-branched alkanoic acid is inhibited by the (R) isomer, although the (R) isomer was not toxic during growth on n-hexadecane. Based on these results, Mcr is suggested to play a critical role in beta-oxidation of methyl-branched alkanes in Mycobacterium. On the other hand, mutant H1 could not grow on n-hexadecane, but it partially retained the ability to grow on pristane. The reduced growth of mutant H1 on pristane suggests that propionyl-CoA is available for cell propagation through the 2-methyl citric acid cycle, since propionyl-CoA is produced through beta-oxidation of pristane.

Crystal structure of Salmonella typhimurium 2-methylisocitrate lyase (PrpB) and its complex with pyruvate and Mg 2+

Propionate metabolism in Salmonella typhimurium occurs via 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (PrpB). Here we report the X-ray crystal structure of the native and the pyruvate/Mg 2+ bound PrpB from S. typhimurium, determined at 2.1 and 2.3 Å, respectively. The structure closely resembles that of the Escherichia coli enzyme. Unlike the E. coli PrpB, Mg 2+ could not be located in the native Salmonella PrpB. Only in pyruvate bound PrpB structure, Mg 2+ was found coordinated with pyruvate. Binding of pyruvate to PrpB seems to induce movement of the Mg 2+ by 2.5 Å from its position found in E. coli native PrpB. In both the native enzyme and pyruvate/Mg 2+ bound forms, the active site loop is completely disordered. Examination of the pocket in which pyruvate and glyoxalate bind to 2-methylisocitrate lyase and isocitrate lyase, respectively, reveals plausible rationale for different substrate specificities of these two enzymes. Structural similarities in substrate and metal atom binding site as well as presence of similar residues in the active site suggest possible similarities in the reaction mechanism.

Residues C123 and D58 of the 2-methylisocitrate lyase (PrpB) enzyme of Salmonella enterica are essential for catalysis.

The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50 degrees C, and the reaction required Mg(2+) ions; equimolar concentrations of Mn(2+) ions were a poor substitute for Mg(2+) (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K(m) of PrpB for 2-MIC was measured at 19 micro M, with a k(cat) of 105 s(-1). Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB(K121A), PrpB(H125A), and PrpB(R122K) mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k(cat) for 2-MIC lyase activity, respectively. The PrpB(D58A) and PrpB(C123A) proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg(2+) ion.

Cloning, expression, purification and preliminary X-ray crystallographic studies of 2-methylisocitrate lyase from Salmonella typhimurium.

In Salmonella typhimurium, propionate is oxidized to pyruvate via the 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (EC 4.1.3.30). Methylisocitrate lyase (molecular weight 32 kDa) with a C-terminal polyhistidine affinity tag has been cloned and overexpressed in Escherichia coli and purified and crystallized under different conditions using the hanging-drop vapour-diffusion technique. Crystals belong to the orthogonal space group P2(1)2(1)2(1), with unit-cell parameters a = 63.600, b = 100.670, c = 204.745 A. A complete data set to 2.5 A resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator.