scholarly journals Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus methylotrophus AS1 Cells Expressing the aroP-Encoded Transporter of Escherichia coli

2009 ◽  
Vol 76 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Yurgis A. V. Yomantas ◽  
Irina L. Tokmakova ◽  
Natalya V. Gorshkova ◽  
Elena G. Abalakina ◽  
Svetlana M. Kazakova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Target Genes ◽  
Aromatic Amino Acids ◽  
Amino Acid Transporter ◽  
Methylotrophic Bacteria ◽  
Methylophilus Methylotrophus ◽  
Acid Transporter

ABSTRACT The isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. We describe a novel method of constructing mutants of the bacterium Methylophilus methylotrophus AS1 that are auxotrophic for aromatic amino acids. The procedure begins with the Mu-driven integration of the Escherichia coli gene aroP, which encodes the common aromatic amino acid transporter, into the genome of M. methylotrophus. The resulting recombinant strain, with improved permeability to certain amino acids and their analogues, was used for mutagenesis. Mutagenesis was carried out by recombinant substitution of the target genes in the chromosome by linear DNA using the FLP-excisable marker flanked with cloned homologous arms longer than 1,000 bp. M. methylotrophus AS1 genes trpE, tyrA, pheA, and aroG were cloned in E. coli, sequenced, disrupted in vitro using a Kmr marker, and electroporated into an aroP carrier recipient strain. This approach led to the construction of a set of marker-less M. methylotrophus AS1 mutants auxotrophic for aromatic amino acids. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.

scholarly journals Cloning, Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

2012 ◽  
Vol 195 (2) ◽  
pp. 340-350 ◽  
Author(s):  
Hein Trip ◽  
Niels L. Mulder ◽  
Juke S. Lolkema
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Lactococcus Lactis ◽  
Aromatic Amino Acids ◽  
Amino Acid Transporter ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
Content Type ◽  
Secondary Amino ◽  
Acid Transporter

ABSTRACTFourteen genes encoding putative secondary amino acid transporters were identified in the genomes ofLactococcus lactissubsp.cremorisstrains MG1363 and SK11 andL. lactissubsp. lactisstrains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake inL. lactiscells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.

scholarly journals Neutral amino acid transporter ASCT2 displays substrate-induced Na+ exchange and a substrate-gated anion conductance

2000 ◽  
Vol 346 (3) ◽  
pp. 705-710 ◽  
Author(s):  
Angelika BRÖER ◽  
Carsten WAGNER ◽  
Florian LANG ◽  
Stefan BRÖER
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Anion Channel ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Xenopus Laevis Oocytes ◽  
Anion Conductance ◽  
Neutral Amino Acid Transporter ◽  
Inward Currents ◽  
Acid Transporter

The neutral amino acid transporter ASCT2 mediates electroneutral obligatory antiport but at the same time requires Na+ for its function. To elucidate the mechanism, ASCT2 was expressed in Xenopus laevis oocytes and transport was analysed by flux studies and two-electrode voltage clamp recordings. Flux studies with 22NaCl indicated that the uptake of one molecule of glutamine or alanine is accompanied by the uptake of four to seven Na+ ions. Similarly to the transport of amino acids, the Na+ uptake was mediated by an obligatory Na+ exchange mechanism that depended on the presence of amino acids but was not stoichiometrically coupled to the amino acid transport. Other cations could not replace Na+ in this transport mechanism. When NaCl was replaced by NaSCN in the transport buffer, the superfusion of oocytes with amino acid substrates resulted in large inward currents, indicating the presence of a substrate-gated anion channel in the ASCT2 transporter. The Km for glutamine derived from these experiments is in good agreement with the Km derived from flux studies; it varied between 40 and 90 μM at holding potentials of -60 and -20 mV respectively. The permeability of the substrate-gated anion conductance decreased in the order SCN- NO3- > I- > Cl- and also required the presence of Na+.

scholarly journals Functional analysis of human aromatic amino acid transporter MCT10/TAT1 using the yeast Saccharomyces cerevisiae

2017 ◽  
Vol 1859 (10) ◽  
pp. 2076-2085 ◽  
Author(s):  
Satoshi Uemura ◽  
Takahiro Mochizuki ◽  
Goyu Kurosaka ◽  
Takanori Hashimoto ◽  
Yuki Masukawa ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Analysis ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Amino Acid Transporter ◽  
Yeast Saccharomyces Cerevisiae ◽  
Acid Transporter

Selective Chemical Deuteration of Aromatic Amino Acids: A Retrospective

1997 ◽  
Author(s):  
K.S. Matthews ◽  
R. Matthews
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Protein Hydrolysate ◽  
Aromatic Amino Acids ◽  
Cellular Protein ◽  
Target Protein ◽  
Chemical Methods ◽  
Lactose Repressor ◽  
Selective Deuteration

In 1970 when we began post-doctoral work in the laboratory of Professor Oleg Jardetzky, selective deuteration of proteins to limit the number of protons present in the system for subsequent analysis was a newly developed and effective technique for NMR exploration of protein structure (Crespi et al., 1968; Markley et al., 1968). This approach allowed more facile assignment of specific resonances and generated the potential to follow the spectroscopic behavior of protons for a specific amino acid sidechain over a broad range of conditions. The primary method for labeling at that time involved growth of microorganisms (generally bacteria or algae) in D2O, followed by isolation of the deuteratedamino acids from a cellular protein hydrolysate. The amino acids isolated were, therefore, completely deuterated. Selective deuteration of a target protein was achieved by growing the producing organism on a mixture of completely deuterated and selected protonated amino acids under conditions that minimized metabolic interconversion of the amino acids. In one-dimensional spectra, aromatic amino acid resonances occur well downfield of the aliphatic resonances, and this region can therefore be examined somewhat independently by utilizing a single protonated aromatic amino acid to simplify the spectrum of the protein. However, the multiple spectral lines generated by aromatic amino acids can be complex and overlapping, precluding unequivocal interpretation. To address this complication, chemical methods were developed to both completely and selectively deuterate side chains of the aromatic amino acids, thereby avoiding the costly necessity of growing large volumes of microorganisms in D2O and subsequent tedious isolation procedures. In addition, selective deuteration of the amino acids simplified the resonance patterns and thereby facilitated assignment and interpretation of spectra. The methods employed were based on exchange phenomena reported in the literature and generated large quantities of material for use in growth of microorganisms for subsequent isolation of selectively labeled protein (Matthews et al., 1977a). The target protein for incorporation of the selectively deuterated aromatic amino acids generated by these chemical methods was the lactose repressor protein from Escherichia coli, and greatly simplified spectra of this 150,000 D protein were produced by this approach.

scholarly journals Intestinal peptidases form functional complexes with the neutral amino acid transporter B0AT1

2012 ◽  
Vol 446 (1) ◽  
pp. 135-148 ◽  
Author(s):  
Stephen J. Fairweather ◽  
Angelika Bröer ◽  
Megan L. O'Mara ◽  
Stefan Bröer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Amino Acid Transporter ◽  
The Body ◽  
Site Directed Mutagenesis ◽  
Substrate Affinity ◽  
Xenopus Laevis Oocytes ◽  
Acid Transporter

The brush-border membrane of the small intestine and kidney proximal tubule are the major sites for the absorption and re-absorption of nutrients in the body respectively. Transport of amino acids is mediated through the action of numerous secondary active transporters. In the mouse, neutral amino acids are transported by B0AT1 [broad neutral (0) amino acid transporter 1; SLC6A19 (solute carrier family 6 member 19)] in the intestine and by B0AT1 and B0AT3 (SLC6A18) in the kidney. Immunoprecipitation and Blue native electrophoresis of intestinal brush-border membrane proteins revealed that B0AT1 forms complexes with two peptidases, APN (aminopeptidase N/CD13) and ACE2 (angiotensin-converting enzyme 2). Physiological characterization of B0AT1 expressed together with these peptidases in Xenopus laevis oocytes revealed that APN increased the substrate affinity of the transporter up to 2.5-fold and also increased its surface expression (Vmax). Peptide competition experiments, in silico modelling and site-directed mutagenesis of APN suggest that the catalytic site of the peptidase is involved in the observed changes of B0AT1 apparent substrate affinity, possibly by increasing the local substrate concentration. These results provide evidence for the existence of B0AT1-containing digestive complexes in the brush-border membrane, interacting differentially with various peptidases, and responding to the dynamic needs of nutrient absorption in the intestine and kidney.

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