Biomedical subjects

Juke S Lolkema

Publications and source records attributed to Juke S Lolkema.

At least 19 recordsLinked to original sources

PubMed2006

Functional characterization and Me ion specificity of a Ca-citrate transporter from Enterococcus faecalis.

Secondary transporters of the bacterial CitMHS family transport citrate in complex with a metal ion. Different members of the family are specific for the metal ion in the complex and have been shown to transport Mg(2+)-citrate, Ca(2+)-citrate or Fe(3+)-citrate. The Fe(3+)-citrate transporter of Streptococcus mutans clusters on the phylogenetic tree on a separate branch with a group of transporters found in the phylum Firmicutes which are believed to be involved in anaerobic citrate degradation. We have cloned and characterized the transporter from Enterococcus faecalis EfCitH in this cluster. The gene was functionally expressed in Escherichia coli and studied using right-side-out membrane vesicles. The transporter catalyzes proton-motive-force-driven uptake of the Ca(2+)-citrate complex with an affinity constant of 3.5 microm. Homologous exchange is catalyzed with a higher efficiency than efflux down a concentration gradient. Analysis of the metal ion specificity of EfCitH activity in right-side-out membrane vesicles revealed a specificity that was highly similar to that of the Bacillus subtilis Ca(2+)-citrate transporter in the same family. In spite of the high sequence identity with the S. mutans Fe(3+)-citrate transporter, no transport activity with Fe(3+) (or Fe(2+)) could be detected. The transporter of E. faecalis catalyzes translocation of citrate in complex with Ca(2+), Sr(2+), Mn(2+), Cd(2+) and Pb(2+) and not with Mg(2+), Zn(2+), Ni(2+) and Co(2+). The specificity appears to correlate with the size of the metal ion in the complex.

Bacterial Proteins↗

PubMed2006

The ntp operon encoding the Na+ V-ATPase of the thermophile Caloramator fervidus.

The V-type ATPase of the thermophile Caloramator fervidus is an ATP-driven Na+ pump. The nucleotide sequence of the ntpFIKECGABD operon containing the structural genes coding for the nine subunits of the enzyme complex was determined. The identity of the proteins in two pairs of subunits (D, E and F, G) that have very similar mobilities on SDS-PAGE of the purified complex (24.3 and 22.7 kDa, and 12.3 and 11.6 kDa) was established by tryptic digestion of the protein bands followed by mass spectrometric analysis of the peptides.

Amino Acid Sequence↗

PubMed2006

The mechanism of the tyrosine transporter TyrP supports a proton motive tyrosine decarboxylation pathway in Lactobacillus brevis.

The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L. brevis was expressed in Lactococcus lactis and functionally characterized using right-side-out membranes. The transporter very efficiently catalyzes homologous tyrosine-tyrosine exchange and heterologous exchange between tyrosine and its decarboxylation product tyramine. Tyrosine-tyramine exchange was shown to be electrogenic. In addition to the exchange mode, the transporter catalyzes tyrosine uniport but at a much lower rate. Analysis of the substrate specificity of the transporter by use of a set of 19 different tyrosine substrate analogues showed that the main interactions between the protein and the substrates involve the amino group and the phenyl ring with the para hydroxyl group. The carboxylate group that is removed in the decarboxylation reaction does not seem to contribute to the affinity of the protein for the substrates significantly. The properties of the TyrP protein are those typical for precursor-product exchangers that operate in proton motive decarboxylation pathways. It is proposed that tyrosine decarboxylation in L. brevis results in proton motive force generation by an indirect proton pumping mechanism.

Amino Acid Transport Systems↗

PubMed2006

Domain structure and pore loops in the 2-hydroxycarboxylate transporter family.

The 2-hydroxycarboxylate transporter (2HCT) family is a family of bacterial secondary transporters for substrates like citrate, malate and lactate. The family is in class ST[3] of the MemGen classification system that groups membrane proteins in structural classes based on hydropathy profile analysis. The combination of computational analysis of the proteins in class ST[3] and available experimental data on members of the 2HCT family has yielded a detailed structural model of the transporters. The core of the model is formed by two homologous domains with opposite orientation in the membrane. Each domain consists of 5 trans membrane segments and contains a pore loop between the 4th and 5th segment. The two pore loops enter the membrane-embedded part from opposite sides of the membrane (trans pore loops) and are believed to form the translocation pathway in the 3D structure. A genome wide study of the cellular location of the C-terminus of all Escherichia coli membrane proteins [Daley et al., 2005. Science 308:1321-1323] showed that the C-termini of the 19 E. coli proteins in class ST[3] were correctly predicted by the structural model.

Bacterial Proteins↗

PubMed2005

Loop VIII/IX of the Na+-citrate transporter CitS of Klebsiella pneumoniae folds into an amphipathic surface helix.

The sodium ion-dependent citrate transporter CitS of Klebsiella pneumoniae is a member of the 2-hydroxycarboxylate transporter (2HCT) family whose members transport divalent citrate in symport with two sodium ions. Profiles of the hydrophobic moment suggested the presence of an amphipathic helical structure in the cytoplasmic loop between transmembrane segments (TMSs) VIII and IX (the AH loop) in all members of the family. Cysteine-scanning mutagenesis was used to study the secondary structure of the AH loop. We have mutated 20 successive residues into cysteine residues, characterized each of the mutants for its transport activity, and determined the accessibility of the residues. Three of the mutants, G324C, F331C, and F332C, had very low citrate transport activity, and two others, I321C and S333C, exhibited significantly decreased activity after treatment of right-side-out membranes with membrane permeable thiol reagent N-ethylmaleimide (NEM), but not with membrane impermeable 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AmdiS) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). No protection against NEM was observed with citrate or sodium ions. Labeling of the cysteine residues in the 20 mutants with the fluorescent probe fluorescein 5-maleimide, in membrane vesicles with an inverted orientation, resulted in a clear periodicity in the accessibility of the residues. Residues expected to be at the hydrophobic face of the putative alpha-helix were not accessible for the label, whereas those at the hydrophilic face were easily accessed and labeled. Pretreatment of whole cells and inside-out membranes expressing the mutants with the membrane impermeable reagent AmdiS confirmed the cytoplasmic localization of the AH region. It is concluded that the loop between TMSs VIII and IX folds into an amphipathic surface helix.

Amino Acid Sequence↗

PubMed2005

Structural and mechanistic diversity of secondary transporters.

Recent reports on the three-dimensional structure of secondary transporters have dramatically increased our knowledge of the translocation mechanism of ions and solutes. The structures of five transporters at atomic resolution have yielded four different folds and as many different translocation mechanisms. The structure of the glutamate transporter homologue GltPh confirmed the role of pore-loop structures as essential parts of the translocation mechanism in one family of secondary transporters. Biochemical evidence for pore-loop structures in several other families suggest that they might be common in secondary transporters, adding to the structural and mechanistic diversity of secondary transporters.

Amino Acid Transport System X-AG↗

PubMed2005

Secondary transporters of the 2HCT family contain two homologous domains with inverted membrane topology and trans re-entrant loops.

The 2-hydroxycarboxylate transporter (2HCT) family of secondary transporters belongs to a much larger structural class of secondary transporters termed ST3 which contains about 2000 transporters in 32 families. The transporters of the 2HCT family are among the best studied in the class. Here we detect weak sequence similarity between the N- and C-terminal halves of the proteins using a sensitive method which uses a database containing the N- and C-terminal halves of all the sequences in ST3 and involves blast searches of each sequence in the database against the whole database. Unrelated families of secondary transporters of the same length and composition were used as controls. The sequence similarity involved major parts of the N- and C-terminal halves and not just a small stretch. The membrane topology of the homologous N- and C-terminal domains was deduced from the experimentally determined topology of the members of the 2HCT family. The domains consist of five transmembrane segments each and have opposite orientations in the membrane. The N terminus of the N-terminal domain is extracellular, while the N terminus of the C-terminal domain is cytoplasmic. The loops between the fourth and fifth transmembrane segment in each domain are well conserved throughout the class and contain a high fraction of residues with small side chains, Gly, Ala and Ser. Experimental work on the citrate transporter CitS in the 2HCT family indicates that the loops are re-entrant or pore loops. The re-entrant loops in the N- and C-terminal domains enter the membrane from opposite sides (trans-re-entrant loops). The combination of inverted membrane topology and trans-re-entrant loops represents a new fold for secondary transporters and resembles the structure of aquaporins and models proposed for Na+/Ca2+ exchangers.

Amino Acid Sequence↗

PubMed2005

Histamine-producing pathway encoded on an unstable plasmid in Lactobacillus hilgardii 0006.

Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.

Base Sequence↗

PubMed2005

The 2-hydroxycarboxylate transporter family: physiology, structure, and mechanism.

The 2-hydroxycarboxylate transporter family is a family of secondary transporters found exclusively in the bacterial kingdom. They function in the metabolism of the di- and tricarboxylates malate and citrate, mostly in fermentative pathways involving decarboxylation of malate or oxaloacetate. These pathways are found in the class Bacillales of the low-CG gram-positive bacteria and in the gamma subdivision of the Proteobacteria. The pathways have evolved into a remarkable diversity in terms of the combinations of enzymes and transporters that built the pathways and of energy conservation mechanisms. The transporter family includes H+ and Na+ symporters and precursor/product exchangers. The proteins consist of a bundle of 11 transmembrane helices formed from two homologous domains containing five transmembrane segments each, plus one additional segment at the N terminus. The two domains have opposite orientations in the membrane and contain a pore-loop or reentrant loop structure between the fourth and fifth transmembrane segments. The two pore-loops enter the membrane from opposite sides and are believed to be part of the translocation site. The binding site is located asymmetrically in the membrane, close to the interface of membrane and cytoplasm. The binding site in the translocation pore is believed to be alternatively exposed to the internal and external media. The proposed structure of the 2HCT transporters is different from any known structure of a membrane protein and represents a new structural class of secondary transporters.

Bacterial Physiological Phenomena↗

PubMed2004

Alternating access and a pore-loop structure in the Na+-citrate transporter CitS of Klebsiella pneumoniae.

CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with 2 Na(+) ions. Reaction of Cys-398 and Cys-414, which are located in a cytoplasmic loop of the protein that is believed to be involved in catalysis, with thiol reagents resulted in significant inhibition of uptake activity. The reactivity of the two residues was determined in single Cys mutants in different catalytic states of the transporter and from both sides of the membrane. The single Cys mutants were shown to have the same transport stoichiometry as wild type CitS, but the C398S mutation was responsible for a 10-fold loss of affinity for Na(+). Both cysteine residues were accessible from the periplasmic as well as from the cytoplasmic side of the membrane by the membrane-impermeable thiol reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) suggesting that the residues are part of the translocation site. Binding of citrate to the outward facing binding site of the transporter resulted in partial protection against inactivation by N-ethylmaleimide, whereas binding to the inward facing binding site resulted in essentially complete protection. A 10-fold higher concentration of citrate was required at the cytoplasmic rather than at the periplasmic side of the membrane to promote protection. Only marginal effects of citrate binding were seen on reactivity with MTSET. Binding of Na(+) at the periplasmic side of the transporter protected both Cys-398 and Cys-414 against reaction with the thiol reagents, whereas binding at the cytoplasmic side was less effective and discriminated between Cys-398 and Cys-414. A model is presented in which part of the cytoplasmic loop containing Cys-398 and Cys-414 folds back into the translocation pore as a pore-loop structure. The loop protrudes into the pore beyond the citrate-binding site that is situated at the membrane-cytoplasm interface.

Bacterial Proteins↗

PubMed2003

Accessibility of cysteine residues in a cytoplasmic loop of CitS of Klebsiella pneumoniae is controlled by the catalytic state of the transporter.

The citrate transporter CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with two sodium ions and one proton. Treatment of CitS with the alkylating agent N-ethylmaleimide resulted in a complete loss of transport activity. Treatment of mutant proteins in which the five endogenous cysteine residues were mutated into serines in different combinations revealed that two cysteine residues located in the C-terminal cytoplasmic loop, Cys-398 and Cys-414, were responsible for the inactivation. Labeling with the membrane impermeable methanethiosulfonate derivatives MTSET and MTSES in right-side-out membrane vesicles showed that the cytoplasmic loop was accessible from the periplasmic side of the membrane. The membrane impermeable but more bulky maleimide AmdiS did not inactivate the transporter in right-side-out membrane vesicles. Inactivation by N-ethylmaleimide, MTSES, and MTSET was prevented by the presence of the co-ion Na(+). Protection was obtained upon binding 2 Na(+), which equals the transport stoichiometry. In the absence of Na(+), the substrate citrate had no effect on the inactivation by permeable or impermeable thiol reagents. In contrast, when subsaturating concentrations of Na(+) were present, citrate significantly reduced inactivation suggesting ordered binding of the substrate and co-ion; citrate is bound after Na(+). In the presence of the proton motive force, the reactivity of the Cys residues was increased significantly for the membrane permeable N-ethylmaleimide, while no difference was observed for the membrane impermeable thiol reagents. The results are discussed in the context of a model for the opening and closing of the translocation pore during turnover of the transporter.

Bacterial Proteins↗

PubMed2003

The A-type ATP synthase subunit K of Methanopyrus kandleri is deduced from its sequence to form a monomeric rotor comprising 13 hairpin domains.

The ntpK gene of the archaeon Methanopyrus kandleri encodes the equivalent of the c subunit of ATP synthase. The gene product contains 1021 residues and consists of 13 homologous domains, each one corresponding to a single helical hairpin. The amino acid sequence of the domains is highly conserved, ranging between 50 and 80% sequence identity. Each of the 13 domains contains a conserved Gln and Glu residue in the N- and C-terminal helix, respectively, both of which are believed to be involved in cation binding. The protein is likely to form the monomeric rotor of the ATP synthase that consists of 13 hairpin domains.

Amino Acid Sequence↗

PubMed2003

Classification of 29 families of secondary transport proteins into a single structural class using hydropathy profile analysis.

A classification scheme for membrane proteins is proposed that clusters families of proteins into structural classes based on hydropathy profile analysis. The averaged hydropathy profiles of protein families are taken as fingerprints of the 3D structure of the proteins and, therefore, are able to detect more distant evolutionary relationships than amino acid sequences. A procedure was developed in which hydropathy profile analysis is used initially as a filter in a BLAST search of the NCBI protein database. The strength of the procedure is demonstrated by the classification of 29 families of secondary transporters into a single structural class, termed ST[3]. An exhaustive search of the database revealed that the 29 families contain 568 unique sequences. The proteins are predominantly from prokaryotic origin and most of the characterized transporters in ST[3] transport organic and inorganic anions and a smaller number are Na(+)/H(+) antiporters. All modes of energy coupling (symport, antiport, uniport) are found in structural class ST[3]. The relevance of the classification for structure/function prediction of uncharacterised transporters in the class is discussed.

Membrane Transport Proteins↗

PubMed2003

A Crh-specific function in carbon catabolite repression in Bacillus subtilis.

Carbon catabolite repression in Bacillus subtilis is mediated by phosphorylation of the phosphoenolpyruvate:carbohydrate phosphotransferase system intermediate HPr at a serine residue catalyzed by HPr kinase. The orthologous protein Crh functions in a similar way, but, unlike HPr, it is not functional in carbohydrate uptake. A specific function for Crh is not known. The role of HPr and Crh in repressing the citM gene encoding the Mg(2+)-citrate transporter was investigated during growth of B. subtilis on different carbon sources. In glucose minimal medium, full repression was supported by both HPr and Crh. Strains deficient in Crh or the regulatory function of HPr revealed the same repression as the wild-type strain. In contrast, in a medium containing succinate and glutamate, repression was specifically mediated via Crh. Repression was relieved in the Crh-deficient strain, but still present in the HPr mutant strain. The data are the first demonstration of a Crh-specific function in B. subtilis and suggest a role for Crh in regulation of expression during growth on substrates other than carbohydrates.

Bacillus subtilis↗

PubMed2003

Conserved residues R420 and Q428 in a cytoplasmic loop of the citrate/malate transporter CimH of Bacillus subtilis are accessible from the external face of the membrane.

CimH of Bacillus subtilis is a secondary transporter for citrate and malate that belongs to the 2-hydroxycarboxylate transporter (2HCT) family. Conserved residues R143, R420, and Q428, located in putative cytoplasmic loops and R432, located at the cytoplasmic end of the C-terminal transmembrane segment XI were mutated to Cys to identify residues involved in binding of the substrates. R143C, R420C, and Q428C revealed kinetics similar to those of the wild-type transporter, while the activity of R432C was reduced by at least 2 orders of magnitude. Conservative replacement of R432 with Lys reduced the activity by 1 order of magnitude, by lowering the affinity for the substrate 10-fold. It is concluded that the arginine residue at position 432 in CimH interacts with one of the carboxylate groups of the substrates. Labeling of the R420C and Q428C mutants with thiol reagents inhibited citrate transport activity. Surprisingly, the cysteine residues in the cytoplasmic loops in both R420C and Q428C were accessible to the small, membrane-impermeable, negatively charged MTSES reagent from the external site of the membrane in a substrate protectable manner. The membrane impermeable reagents MTSET,(1) which is positively charged, and AMdiS, which is negatively charged like MTSES but more bulky, did not inhibit R420C and Q428C. It is suggested that the access pathway is optimized for small, negatively charged substrates. Either the cytoplasmic loop containing residues R420 and Q428 is partly protruding to the outside, possibly in a reentrant loop like structure, or alternatively, a water-filled substrate translocation pathway extents to the cytoplasm-membrane interface.

Arginine↗

PubMed2003

Transporters involved in uptake of di- and tricarboxylates in Bacillus subtilis.

Di- and tricarboxylates found as intermediates in the tricarboxylic acid cycle can be utilized by many bacteria and serve as carbon and energy source under aerobic and anaerobic conditions. A prerequisite for metabolism is that the carboxylates are transported into the cells across the cytoplasmic membrane. Bacillus subtilis is able to metabolize many di- and tricarboxylates and in this overview the available data on all known and putative di- and tricarboxylate transporters in B. subtilis is summarized. The B. subtilis transporters, that are of the secondary type, are discussed in the context of the protein families to which they belong. Available data on biochemical characterization, regulation of gene expression and the physiological function is summarized. It is concluded that in B. subtilis multiple transporters are present for tricarboxylic acid cycle intermediates.

Bacillus subtilis↗

PubMed2003

Subunit composition, structure, and distribution of bacterial V-type ATPases.

The overall structure of V-ATPase complexes resembles that of F-type ATPases, but the stalk region is different and more complex. Database searches followed by sequence analysis of the five water-soluble stalk region subunits C-G revealed that (i) to date V-ATPases are found in 16 bacterial species, (ii) bacterial V-ATPases are closer to archaeal A-ATPases than to eukaryotic V-ATPases, and (iii) different groups of bacterial V-ATPases exist. Inconsistencies in the nomenclature of types and subunits are addressed. Attempts to assign subunit positions in V-ATPases based on biochemical experiments, chemical cross-linking, and electron microscopy are discussed. A structural model for prokaryotic and eukaryotic V-ATPases is proposed. The prokaryotic V-ATPase is considered to have a central stalk between headpiece and membrane flanked by two peripheral stalks. The eukaryotic V-ATPases have one additional peripheral stalk.

Bacterial Proteins↗

PubMed2003

CcpA-independent regulation of expression of the Mg2+ -citrate transporter gene citM by arginine metabolism in Bacillus subtilis.

Transcriptional regulation of the Mg(2+)-citrate transporter, CitM, the main citrate uptake system of Bacillus subtilis, was studied during growth in rich medium. Citrate in the growth medium was required for induction under all growth conditions. In Luria-Bertani medium containing citrate, citM expression was completely repressed during the exponential growth phase, marginally expressed in the transition phase, and highly expressed in the stationary growth phase. The repression was relieved when the cells were grown in spent Luria-Bertani medium. The addition of a mixture of 18 amino acids restored repression. L-Arginine in the mixture appeared to be solely responsible for the repression, and ornithine appeared to be an equally potent repressor of citM expression. Studies of mutant strains deficient in RocR and SigL, proteins required for the expression of the enzymes of the arginase pathway, confirmed that uptake into the cell and, most likely, conversion of arginine to ornithine were required for repression. Arginine-mediated repression was independent of a functional CcpA, the global regulator protein in carbon catabolite repression (CCR). Nevertheless, CCR-mediated repression was the major mechanism controlling the expression during exponential growth, while the newly described, CcpA-independent arginine-mediated repression was specifically apparent during the transition phase of growth.

Arginine↗