Tricarboxylate Transporter Research Articles

Regulation of tricarboxylate transport and metabolism in Acinetobacter baylyi ADP1.

Despite the significant presence of plant-derived tricarboxylic acids in some environments, few studies detail the bacterial metabolism of trans-aconitic acid (Taa) and tricarballylic acid (Tcb). In a soil bacterium, Acinetobacter baylyi ADP1, we discovered interrelated pathways for the consumption of Taa and Tcb. An intricate regulatory scheme tightly controls the transport and catabolism of both compounds and may reflect that they can be toxic inhibitors of the tricarboxylic acid cycle. The genes encoding two similar LysR-type transcriptional regulators, TcuR and TclR, were clustered on the chromosome with tcuA and tcuB, genes required for Tcb consumption. The genetic organization differed from that in Salmonella enterica serovar Typhimurium, in which tcuA and tcuB form an operon with a transporter gene, tcuC. In A. baylyi, tcuC was not cotranscribed with tcuAB. Rather, tcuC was cotranscribed with a gene, designated pacI, encoding an isomerase needed for Taa consumption. TcuC appears to transport Tcb and cis-aconitic acid (Caa), the presumed product of PacI-mediated periplasmic isomerization of Taa. Two operons, tcuC-pacI and tcuAB, were transcriptionally controlled by both TcuR and TclR, which have overlapping functions. We investigated the roles of the two regulators in activating transcription of both operons in response to multiple effector compounds, including Taa, Tcb, and Caa.IMPORTANCEIngestion of Taa and Tcb by grazing livestock can cause a serious metabolic disorder called grass tetany. The disorder, which results from Tcb absorption by ruminants, focuses attention on the metabolism of tricarboxylic acids. Additional interest stems from efforts to produce tricarboxylic acids as commodity chemicals. Improved understanding of bacterial enzymes and pathways for tricarboxylic acid metabolism may contribute to new biomanufacturing strategies.

Role of sodium dependent SLC13 transporter inhibitors in various metabolic disorders.

The sodium dependent SLC13 family transporters comprise of five genes SLC13A1, SLC13A2 (NaDC1), SLC13A3 (NaDC3), SLC13A4 and SLC13A5 (NaCT). Among them, NaDC1, NaDC3 and NaCT are sodium dependent transporters belonging to family of dicarboxylates (succinate, malate, α-ketoglutarate) and tricarboxylates (citrate). The mouse and the human NaCT structures have still not been crystallized, therefore structural information is taken from the related bacterial transporter of VcINDY. Citrate in the cytosol works as a precursor for the fatty acid synthesis, cholesterol, and low-density lipoproteins. The excess citrate from the matrix is translocated to the cytosol for fatty acid synthesis through these transporters and thus controls the energy balance by downregulating the glycolysis, tricarboxylic acid (TCA), and fatty acid breakdown. These transporters play an important role in regulating various metabolic diseases including cancer, diabetes, obesity, fatty liver diseases and CNS disorders. These di and tricarboxylate transporters are emerging as new targets for metabolic disorders such as obesity and diabetes. The mutation in the function of the NaCT causes several neurological diseases including neonatal epilepsy and impaired brain development whereas mutation of genes coding for citrate transport present in the liver may provide positive effect. Therefore, continued efforts from the earlier work on citrate transporters are required for the development of citrate inhibitors. This review discusses the structure, function, and regulation of the NaCT transporter. The review also highlights citrate role in diagnosing diseases such as cancer, diabetes, fatty liver, and diabetes. The therapeutic perspective of synthetic inhibitors against NaCT transporters is succinctly summarized.

Tricarboxylate Citrate Transporter of an Oleaginous Fungus Mucor circinelloides WJ11: From Function to Structure and Role in Lipid Production.

The citrate transporter protein (CTP) plays an important role in citrate efflux from the mitochondrial matrix to cytosol that has great importance in oleaginous fungi. The cytoplasmic citrate produced after citrate efflux serves as the primary carbon source for the triacylglycerol and cholesterol biosynthetic pathways. Because of the CTP's importance, our laboratory has extensively studied its structure/function relationships in Mucor circinelloides to comprehend its molecular mechanism. In the present study, the tricarboxylate citrate transporter (Tct) of M. circinelloides WJ11 has been cloned, overexpressed, purified, kinetically, and structurally characterized. The Tct protein of WJ11 was expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system for kinetic studies. Our results showed that Tct has a high affinity for citrate with Km 0.018 mM. Furthermore, the tct overexpression and knockout plasmids were created and transformed into M. circinelloides WJ11. The mitochondria of the tct-overexpressing transformant of M. circinelloides WJ11 showed a 49% increase in citrate efflux, whereas the mitochondria of the tct-knockout transformant showed a 39% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. To elucidate the structure-function relationship of this biologically important transporter a 3D model of the mitochondrial Tct protein was constructed using homology modeling. The overall structure of the protein is V-shaped and its 3D structure is dimeric. The transport stability of the structure was also assessed by molecular dynamics simulation studies. The activity domain was identified to form hydrogen bond and stacking interaction with citrate and malate upon docking. Tricarboxylate citrate transporter has shown high binding energy of −4.87 kcal/mol to citric acid, while −3.80 kcal/mol to malic acid. This is the first report of unraveling the structural characteristics of WJ11 mitochondrial Tct protein and understanding the approach of the transporting toward its substrate. In conclusion, the present findings support our efforts to combine functional and structural data to better understand the Tct of M. circinelloides at the molecular level and its role in lipid accumulation.

Characterization of JEN family carboxylate transporters from the acid-tolerant yeast Pichia kudriavzevii and their applications in succinic acid production.

SummaryThe unconventional yeast Pichia kudriavzevii is renowned for its ability to survive at low pH and has been exploited for the industrial production of various organic acids, especially succinic acid (SA). However, P. kudriavzevii can also utilize the di‐ and tricarboxylate intermediates of the Krebs cycle as the sole carbon sources for cell growth, which may adversely affect the extracellular accumulation of SA. Because the carboxylic acid transport machinery of P. kudriavzevii remains poorly understood, here, we focused on studying its SA transportation process from the perspective of mining and characterization of dicarboxylate transporters in a newly isolated acid‐tolerant P. kudriavzevii strain CY902. Through genome sequencing and transcriptome analysis, two JEN family carboxylate transporters (PkJEN2‐1 and PkJEN2‐2) were found to be involved in SA transport. Substrate specificity analysis revealed that both PkJEN proteins are active dicarboxylate transporters, that can effectively import succinate, fumarate and L‐malate into the cell. In addition, PkJEN2‐1 can transport α‐ketoglutarate, while PkJEN2‐2 cannot. Since PkJEN2‐1 shows higher transcript abundance than PkJEN2‐2, its role in dicarboxylate transport is more important than PkJEN2‐2. In addition, PKJEN2‐2 is also responsible for the uptake of citrate. To our best knowledge, this is the first study to show that a JEN2 subfamily transporter is involved in tricarboxylate transport in yeast. A combination of model‐based structure analysis and rational mutagenesis further proved that amino acid residues 392‐403 of the tenth transmembrane span (TMS‐X) of PkJEN2‐2 play an important role in determining the specificity of the tricarboxylate substrate. Moreover, these two PkJEN transporters only exhibited inward transport activity for SA, and simultaneous inactivation of both PkJEN transporters reduced the SA influx, resulting in enhanced extracellular accumulation of SA in the late stage of fermentation. This work provides useful information on the mechanism of di‐/tricarboxylic acid utilization in P. kudriavzevii, which will help improve the organic acid production performance of this microbial chassis.

An Optimized Ustilago maydis for Itaconic Acid Production at Maximal Theoretical Yield.

Ustilago maydis, a member of the Ustilaginaceae family, is a promising host for the production of several metabolites including itaconic acid. This dicarboxylate has great potential as a bio-based building block in the polymer industry, and is of special interest for pharmaceutical applications. Several itaconate overproducing Ustilago strains have been generated by metabolic and morphology engineering. This yielded stabilized unicellular morphology through fuz7 deletion, reduction of by-product formation through deletion of genes responsible for itaconate oxidation and (glyco)lipid production, and the overexpression of the regulator of the itaconate cluster ria1 and the mitochondrial tricarboxylate transporter encoded by mttA from Aspergillus terreus. In this study, itaconate production was further optimized by consolidating these different optimizations into one strain. The combined modifications resulted in itaconic acid production at theoretical maximal yield, which was achieved under biotechnologically relevant fed-batch fermentations with continuous feed.

Multiple Genes of Symbiotic Plasmid and Chromosome in Type II Peanut Bradyrhizobium Strains Corresponding to the Incompatible Symbiosis With Vigna radiata.

Rhizobia are capable of establishing compatible symbiosis with their hosts of origin and plants in the cross-nodulation group that the hosts of origin belonged to. However, different from the normal peanut Bradyrhizobium (Type I strains), the Type II strains showed incompatible symbiosis with Vigna radiata. Here, we employed transposon mutagenesis to identify the genetic loci related to this incompatibility in Type II strain CCBAU 53363. As results, seven Tn5 transposon insertion mutants resulted in an increase in nodule number on V. radiata. By sequencing analysis of the sequence flanking Tn5 insertion, six mutants were located in the chromosome of CCBAU 53363, respectively encoding acyltransferase (L265) and hypothetical protein (L615)—unique to CCBAU 53363, two hypothetical proteins (L4 and L82), tripartite tricarboxylate transporter substrate binding protein (L373), and sulfur oxidation c-type cytochrome SoxA (L646), while one mutant was in symbiotic plasmid encoding alanine dehydrogenase (L147). Significant differences were observed in L147 gene sequences and the deduced protein 3D structures between the Type II (in symbiotic plasmid) and Type I strains (in chromosome). Conversely, strains in both types shared high homologies in the chromosome genes L373 and L646 and in their protein 3D structures. These data indicated that the symbiotic plasmid gene in Type II strains might have directly affected their symbiosis incompatibility, whereas the chromosome genes might be indirectly involved in this process by regulating the plasmid symbiosis genes. The seven genes may initially explain the complication associated with symbiotic incompatibility.

Metabolic engineering of an acid-tolerant yeast strain Pichia kudriavzevii for itaconic acid production

Itaconic acid (IA), or 2-methylenesuccinic acid, has a broad spectrum of applications in the biopolymer industry owing to the presence of one vinyl bond and two acid groups in the structure. Its polymerization can follow a similar mechanism as acrylic acid but additional functionality can be incorporated into the extra beta acid group. Currently, the bio-based production of IA in industry relies on the fermentation of the filamentous fungus Aspergillus terreus. However, the difficulties associated with the fermentation undertaken by filamentous fungi together with the pathogenic potential of A. terreus pose a serious challenge for industrial-scale production. In recent years, there has been increasing interest in developing alternative production hosts for fermentation processes that are more homogenous in the production of organic acids.Pichia kudriavzevii is a non-conventional yeast with high acid tolerance to organic acids at low pH, which is a highly desirable trait by easing downstream processing. We introduced cis-aconitic acid decarboxylase gene (cad) from A. terreus (designated At_cad) into this yeast and established the initial titer of IA at 135 ​± ​5 ​mg/L. Subsequent overexpression of a native mitochondrial tricarboxylate transporter (herein designated Pk_mttA) presumably delivered cis-aconitate efficiently to the cytosol and doubled the IA production. By introducing the newly invented CRISPR-Cas9 system into P. kudriavzevii, we successfully knocked out both copies of the gene encoding isocitrate dehydrogenase (ICD), aiming to increase the availability of cis-aconitate. The resulting P. kudriavzevii strain, devoid of ICD and overexpressing Pk_mttA and At_cad on its genome produced IA at 505 ​± ​17.7 ​mg/L in shake flasks, and 1232 ​± ​64 ​mg/L in fed-batch fermentation. Because the usage of an acid-tolerant species does not require pH adjustment during fermentation, this work demonstrates the great potential of engineering P. kudriavzevii as an industrial chassis for the production of organic acid.

A universal stress protein in Mycobacterium smegmatis sequesters the cAMP-regulated lysine acyltransferase and is essential for biofilm formation

Universal stress proteins (USPs) are present in many bacteria, and their expression is enhanced under various environmental stresses. We have previously identified a USP in Mycobacterium smegmatis that is a product of the msmeg_4207 gene and is a substrate for a cAMP-regulated protein lysine acyltransferase (KATms; MSMEG_5458). Here, we explored the role of this USP (USP4207) in M. smegmatis and found that its gene is present in an operon that also contains genes predicted to encode a putative tripartite tricarboxylate transporter (TTT). Transcription of the TTT-usp4207 operon was induced in the presence of citrate and tartrate, perhaps by the activity of a divergent histidine kinase-response regulator gene pair. A usp4207-deleted strain had rough colony morphology and reduced biofilm formation compared with the WT strain; however, both normal colony morphology and biofilm formation were restored in a Δusp4207Δkatms strain. We identified several proteins whose acetylation was lost in the Δkatms strain, and whose transcript levels increased in M. smegmatis biofilms along with that of USP4207, suggesting that USP4207 insulates KATms from its other substrates in the cell. We propose that USP4207 sequesters KATms from diverse substrates whose activities are down-regulated by acylation but are required for biofilm formation, thus providing a defined role for this USP in mycobacterial physiology and stress responses.

A tripartite tricarboxylate transporter (MIM_c39170-MIM_c39210) of Advenella mimigardefordensis DPN7T is involved in citrate uptake.

To date, tripartite tricarboxylate transport (TTT) systems are not well characterized in most organisms. To investigate which carbon sources are transported by the TTT system of A. mimigardefordensis DPN7T, single deletion mutants were generated lacking either completely both sets of genes encoding for these transport systems tctABCDE1 and tctABDE2 in the organism or the two genes encoding for the regulatory components of the third chosen TTT system, tctDE3. Deletion of tctABCDE1 (MIM_c39170-MIM_c39210) in Advenella mimigardefordensis strain DPN7T led to inhibition of growth of the cells with citrate indicating that TctABCDE1 is the transport system for the uptake of citrate. Because of the negative phenotype, it was concluded that this deletion cannot be substituted by other transporters encoded in the genome of strain DPN7T. A triple deletion mutant of A. mimigardefordensis lacking both complete TTT transport systems and the regulatory components of the third chosen system (ΔTctABCDE1 ΔTctABDE2 ΔTctDE3) showed a leaky growth with α-ketoglutarate in comparison with the wild type. The other investigated TTT (TctABDE3, MIM_c17190-MIM_c17220) is most probably involved in the transport of α-ketoglutarate. Additionally, thermoshift assays with TctC1 (MIM_c39190) showed a significant shift in the melting temperature of the protein in the presence of citrate whereas no shift occurred with α-ketoglutarate. A dissociation constant Kd for citrate of 41.7μM was determined. Furthermore, alternative α-ketoglutarate transport was investigated via in silico analysis.

The interplay between transport and metabolism in fungal itaconic acid production

Besides enzymatic conversions, many eukaryotic metabolic pathways also involve transport proteins that shuttle molecules between subcellular compartments, or into the extracellular space. Fungal itaconate production involves two such transport steps, involving an itaconate transport protein (Itp), and a mitochondrial tricarboxylate transporter (Mtt). The filamentous ascomycete Aspergillus terreus and the unicellular basidiomycete Ustilago maydis both produce itaconate, but do so via very different molecular pathways, and under very different cultivation conditions. In contrast, the transport proteins of these two strains are assumed to have a similar function. This study aims to investigate the roles of both the extracellular and mitochondrial transporters from these two organisms by expressing them in the corresponding U. maydis knockouts and monitoring the extracellular product concentrations. Both transporters from A. terreus complemented their corresponding U. maydis knockouts in mediating itaconate production. Surprisingly, complementation with At_MfsA from A. terreus led to a partial switch from itaconate to (S)-2-hydroxyparaconate secretion. Apparently, the export protein from A. terreus has a higher affinity for (S)-2-hydroxyparaconate than for itaconate, even though this species is classically regarded as an itaconate producer. Complementation with At_MttA increased itaconate production by 2.3-fold compared to complementation with Um_Mtt1, indicating that the mitochondrial carrier from A. terreus supports a higher metabolic flux of itaconic acid precursors than its U. maydis counterpart. The biochemical implications of these differences are discussed in the context of the biotechnological application in U. maydis and A. terreus for the production of itaconate and (S)-2-hydroxyparaconate.

A New Mechanism for High-Affinity Uptake of C4-Dicarboxylates in Bacteria Revealed by the Structure of Rhodopseudomonas palustris MatC (RPA3494), a Periplasmic Binding Protein of the Tripartite Tricarboxylate Transporter (TTT) Family

C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric acid cycle, while in anaerobic bacteria, they are important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes, but only one of these utilizes an extracytoplasmic solute binding protein to achieve high-affinity uptake. Here, we identify the MatBAC system from the photosynthetic bacterium Rhodopseudomonas palustris as the first member of the tripartite tricarboxylate transport family to be involved in C4-dicarboxylate transport. Tryptophan fluorescence spectroscopy showed that MatC, the periplasmic binding protein from this system, binds to l- and d-malate with Kd values of 27 and 21 nM, respectively, the highest reported affinity to date for these C4-dicarboxylates, and to succinate (Kd = 110 nM) and fumarate (Kd = 400 nM). The 2.1-Å crystal structure of MatC with bound malate shows a high level of substrate coordination, with participation of two water molecules that bridge hydrogen bonds between the ligand proximal carboxylic group and the main chain of two conserved loops in the protein structure. The substrate coordination in MatC correlates with the binding data and explains the protein's selectivity for different substrates and respective binding affinities. Our results reveal a new function in C4-dicarboxylate transport by members of the poorly characterized tripartite tricarboxylate transport family, which are widely distributed in bacterial genomes but for which details of structure–function relationships and transport mechanisms have been lacking.

Enhanced synthesis of medium-chain-length poly(3-hydroxyalkanoates) by inactivating the tricarboxylate transport system of Pseudomonas putida KT2440 and process development using waste vegetable oil

Abstract The use of waste materials as feedstock for biosynthesis of valuable compounds has been an intensive area of research aiming at diminishing the consumption of non-renewable materials. In this study, P. putida KT2440 was employed as a cell factory for the bioconversion of waste vegetable oil into medium-chain-length Polyhydroxyalkanoates. In the presence of the waste oil this environmental strain is capable of secreting enzymes with lipase activities that enhance the bioavailability of this hydrophobic carbon substrate. It was also found that the oxygen transfer coefficient is directly correlated with high PHA levels in KT2440 cells when metabolizing the waste frying oil. By knocking out the tctA gene, encoding for an enzyme of the tripartite carboxylate transport system, an enhanced intracellular level of mcl-PHA was found in the engineered strain when grown on fatty acids. Batch bioreactors showed that the KT2440 strain produced 1.01 (g⋅L−1) of PHA whereas the engineered ΔtctA P. putida strain synthesized 1.91 (g⋅L−1) after 72 h cultivation on 20 (g⋅L−1) of waste oil, resulting in a nearly 2-fold increment in the PHA volumetric productivity. Taken together, this work contributes to accelerate the pace of development for efficient bioconversion of waste vegetable oils into sustainable biopolymers.

Mammalian Solute Carrier (SLC)-like transporters of Legionella pneumophila

Acquisition of nutrients during intra-vacuolar growth of L. pneumophila within macrophages or amoebae is poorly understood. Since many genes of L. pneumophila are acquired by inter-kingdom horizontal gene transfer from eukaryotic hosts, we examined the presence of human solute carrier (SLC)-like transporters in the L. pneumophila genome using I-TASSER to assess structural alignments. We identified 11 SLC-like putative transporters in L. pneumophila that are structurally similar to SLCs, eight of which are amino acid transporters, and one is a tricarboxylate transporter. The two other transporters, LstA and LstB, are structurally similar to the human glucose transporter, SLC2a1/Glut1. Single mutants of lstA or lstB have decreased ability to import, while the lstA/lstB double mutant is severely defective for uptake of glucose. While lstA or lstB single mutants are not defective in intracellular proliferation within Acanthamoeba polyphaga and human monocyte-derived macrophages, the lstA/lstB double mutant is severely defective in both host cells. The two phenotypic defects of the lstA/lstB double mutant in uptake of glucose and intracellular replication are both restored upon complementation of either lstA or lstB. Our data show that the two glucose transporters, LstA and LstB, are redundant and are required for intracellular replication within human macrophages and amoebae.

Massive over-representation of solute-binding proteins (SBPs) from the tripartite tricarboxylate transporter (TTT) family in the genome of the α-proteobacterium Rhodoplanes sp. Z2-YC6860.

Lineage-specific expansion (LSE) of protein families is a widespread phenomenon in many eukaryotic genomes, but is generally more limited in bacterial genomes. Here, we report the presence of 434 genes encoding solute-binding proteins (SBPs) from the tripartite tricarboxylate transporter (TTT) family, within the 8.2 Mb genome of the α-proteobacterium Rhodoplanes sp. Z2-YC6860, a gene family over-representation of unprecedented abundance in prokaryotes. Representing over 6 % of the total number of coding sequences, the SBP genes are distributed across the whole genome but are found rarely in low-GC islands, where the gene density for this family is much lower. This observation, and the much higher sequence identity between the 434 Rhodoplanes TTT SBPs compared with the average identity between homologues from different species, is indicative of a key role for LSE in the expansion. The TTT SBP genes were found in the vicinity of genes encoding membrane components of transport systems from different families, as well as regulatory proteins such as histidine-kinases and transcription factors, indicating a broad range of functions around the sensing, response and transport of organic compounds. A smaller expansion of TTT SBPs is known in some species of the β-proteobacteria Bordetella and we observed similar expansions in other β-proteobacterial lineages, including members of the genus Comamonas and the industrial biotechnology organism Cupriavidus necator, indicating that strong environmental selection can drive SBP duplication and specialisation from multiple evolutionary starting points.

Tripartite ATP-Independent Periplasmic (TRAP) Transporters and Tripartite Tricarboxylate Transporters (TTT): From Uptake to Pathogenicity.

The ability to efficiently scavenge nutrients in the host is essential for the viability of any pathogen. All catabolic pathways must begin with the transport of substrate from the environment through the cytoplasmic membrane, a role executed by membrane transporters. Although several classes of cytoplasmic membrane transporters are described, high-affinity uptake of substrates occurs through Solute Binding-Protein (SBP) dependent systems. Three families of SBP dependant transporters are known; the primary ATP-binding cassette (ABC) transporters, and the secondary Tripartite ATP-independent periplasmic (TRAP) transporters and Tripartite Tricarboxylate Transporters (TTT). Far less well understood than the ABC family, the TRAP transporters are found to be abundant among bacteria from marine environments, and the TTT transporters are the most abundant family of proteins in many species of β-proteobacteria. In this review, recent knowledge about these families is covered, with emphasis on their physiological and structural mechanisms, relating to several examples of relevant uptake systems in pathogenicity and colonization, using the SiaPQM sialic acid uptake system from Haemophilus influenzae and the TctCBA citrate uptake system of Salmonella typhimurium as the prototypes for the TRAP and TTT transporters, respectively. High-throughput analysis of SBPs has recently expanded considerably the range of putative substrates known for TRAP transporters, while the repertoire for the TTT family has yet to be fully explored but both types of systems most commonly transport carboxylates. Specialized spectroscopic techniques and site-directed mutagenesis have enriched our knowledge of the way TRAP binding proteins capture their substrate, while structural comparisons show conserved regions for substrate coordination in both families. Genomic and protein sequence analyses show TTT SBP genes are strikingly overrepresented in some bacteria, especially in the β-proteobacteria and some α-proteobacteria. The reasons for this are not clear but might be related to a role for these proteins in signaling rather than transport.

Structural basis for high‐affinity adipate binding to AdpC (RPA4515), an orphan periplasmic‐binding protein from the tripartite tricarboxylate transporter (TTT) family in Rhodopseudomonas palustris

Protein structure co-ordinates are available in the PDB under the accession numbers 5OEI and 5OKU.

Sequence-Based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families.

Arylmalonate Decarboxylases (AMDases, EC 4.1.1.76) are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta-, and Gamma-proteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the tripartite tricarboxylate transporters family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99%) of the (R)-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes.

Prevention of diet-induced hepatic steatosis and hepatic insulin resistance by second generation antisense oligonucleotides targeted to the longevity gene mIndy (Slc13a5).

Reducing the expression of the Indy (I'm Not Dead Yet) gene in lower organisms extends life span by mechanisms resembling caloric restriction. Similarly, deletion of the mammalian homolog, mIndy (Slc13a5), encoding for a plasma membrane tricarboxylate transporter, protects from aging- and diet-induced adiposity and insulin resistance in mice. The organ specific contribution to this phenotype is unknown. We examined the impact of selective inducible hepatic knockdown of mIndy on whole body lipid and glucose metabolism using 2'-O-methoxyethyl chimeric anti-sense oligonucleotides (ASOs) in high-fat fed rats. 4-week treatment with 2'-O-methoxyethyl chimeric ASO reduced mIndy mRNA expression by 91% (P=0.001) compared to control ASO. Besides similar body weights between both groups, mIndy-ASO treatment lead to a 74% reduction in fasting plasma insulin concentrations as well as a 35% reduction in plasma triglycerides. Moreover, hepatic triglyceride content was significantly reduced by the knockdown of mIndy, likely mediating a trend to decreased basal rates of endogenous glucose production as well as an increased suppression of hepatic glucose production by 25% during a hyperinsulinemic-euglycemic clamp. Together, these data suggest that inducible liver-selective reduction of mIndy in rats is able to ameliorate hepatic steatosis and insulin resistance, conditions occurring with high calorie diets and during aging.

Determinants of Substrate and Cation Transport in the Human Na+/Dicarboxylate Cotransporter NaDC3

Metabolic intermediates, such as succinate and citrate, regulate important processes ranging from energy metabolism to fatty acid synthesis. Cytosolic concentrations of these metabolites are controlled, in part, by members of the SLC13 gene family. The molecular mechanism underlying Na(+)-coupled di- and tricarboxylate transport by this family is understood poorly. The human Na(+)/dicarboxylate cotransporter NaDC3 (SLC13A3) is found in various tissues, including the kidney, liver, and brain. In addition to citric acid cycle intermediates such as α-ketoglutarate and succinate, NaDC3 transports other compounds into cells, including N-acetyl aspartate, mercaptosuccinate, and glutathione, in keeping with its dual roles in cell nutrition and detoxification. In this study, we construct a homology structural model of NaDC3 on the basis of the structure of the Vibrio cholerae homolog vcINDY. Our computations are followed by experimental testing of the predicted NaDC3 structure and mode of interaction with various substrates. The results of this study show that the substrate and cation binding domains of NaDC3 are composed of residues in the opposing hairpin loops and unwound portions of adjacent helices. Furthermore, these results provide a possible explanation for the differential substrate specificity among dicarboxylate transporters that underpin their diverse biological roles in metabolism and detoxification. The structural model of NaDC3 provides a framework for understanding substrate selectivity and the Na(+)-coupled anion transport mechanism by the human SLC13 family and other key solute carrier transporters.

Unravelling the complete genome sequence of Advenella mimigardefordensis strain DPN7T and novel insights in the catabolism of the xenobiotic polythioester precursor 3,3′-dithiodipropionate

Advenella mimigardefordensis strain DPN7(T) is a remarkable betaproteobacterium because of its extraordinary ability to use the synthetic disulfide 3,3'-dithiodipropionic acid (DTDP) as the sole carbon source and electron donor for aerobic growth. One application of DTDP is as a precursor substrate for biotechnically synthesized polythioesters (PTEs), which are interesting non-degradable biopolymers applicable for plastics materials. Metabolic engineering for optimization of PTE production requires an understanding of DTDP conversion. The genome of A. mimigardefordensis strain DPN7(T) was sequenced and annotated. The circular chromosome was found to be composed of 4,740,516 bp and 4112 predicted ORFs, whereas the circular plasmid consisted of 23,610 bp and 24 predicted ORFs. The genes participating in DTDP catabolism had been characterized in detail previously, but knowing the complete genome sequence and with support of Tn5: :mob-induced mutants, putatively involved transporter proteins and a transcriptional regulator were also identified. Most probably, DTDP is transported into the cell by a specific tripartite tricarboxylate transport system and is then cleaved by the disulfide reductase LpdA, sulfoxygenated by the 3-mercaptopropionate dioxygenase Mdo, activated by the CoA ligase SucCD and desulfinated by the acyl-CoA dehydrogenase-like desulfinase AcdA. Regulation of this pathway is presumably performed by a transcriptional regulator of the xenobiotic response element family. The excessive sulfate that is inevitably produced is secreted by the cells by a unique sulfate exporter of the CPA (cation : proton antiporter) superfamily.