N1-aminopropylagmatine, a new polyamine produced as a key intermediate in polyamine biosynthesis of an extreme thermophile, Thermus thermophilus.
ABSTRACT In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase = agmatine ureohydrolase) accumulated N1-aminopropylagmatine (N8-amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N1-aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N1-Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N1-Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N1-Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N1-aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N1-aminopropylagmatine to spermidine but does not act on agmatine.
- SourceAvailable from: Alejandro Maass[Show abstract] [Hide abstract]
ABSTRACT: In this study, we present the first metabolic profiles for two bioleaching bacteria using capillary electrophoresis coupled with mass spectrometry. The bacteria, Acidithiobacillus ferrooxidans strain Wenelen (DSM 16786) and Acidithiobacillus thiooxidans strain Licanantay (DSM 17318), were sampled at different growth phases and on different substrates: the former was grown with iron and sulfur, and the latter with sulfur and chalcopyrite. Metabolic profiles were scored from planktonic and sessile states. Spermidine was detected in intra- and extracellular samples for both strains, suggesting it has an important role in biofilm formation in the presence of solid substrate. The canonical pathway for spermidine synthesis seems absent as its upstream precursor, putrescine, was not present in samples. Glutathione, a catalytic activator of elemental sulfur, was identified as one of the most abundant metabolites in the intracellular space in A. thiooxidans strain Licanantay, confirming its participation in the sulfur oxidation pathway. Amino acid profiles varied according to the growth conditions and bioleaching species. Glutamic and aspartic acid were highly abundant in intra- and extracellular extracts. Both are constituents of the extracellular matrix, and have a probable role in cell detoxification. This novel metabolomic information validates previous knowledge from in silico metabolic reconstructions based on genomic sequences, and reveals important biomining functions such as biofilm formation, energy management and stress responses. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-012-0443-3) contains supplementary material, which is available to authorized users.Metabolomics 02/2013; 9(1):247-257. · 4.43 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Longer/branched-chain polyamines are unique polycations found in thermophiles. N(4)-aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N(4)-bis(aminopropyl)spermidine, an isomer of N(4)-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by HPLC, GC, and GC-MS. N(4)-bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N(4)-bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by LIT-TOFMS with ESI-trap instrument following analysis by MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified and their N(4)-bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N(4)-bis(aminopropyl)spermidine from spermidine via N(4)-aminopropylspermidine. Compared with wild-type, the bpsA disrupted strain DBP1 grew at 85°C with a slightly longer lag phase, but was unable to grow at 93°C. HPLC analysis showed that both N(4)-aminopropylspermidine and N(4)-bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison with orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high temperature environments.Journal of bacteriology 03/2014; · 3.94 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Biogenic amines (BA; histamine, tyramine, tryptamine, phenylethylamine, cadaverine) and polyamines (PA; putrescine, spermidine, spermine) content was determined in Dutch-type semi-hard cheese (ripening 176days) using an experimental design two producers×two starter cultures×two fat contents. Content of quantitatively most important BA (tyramine and histamine) and PA (putrescine) in the cheese samples at the end of ripening varied in the range 5–392 and 22–59, and 1–132mgkg−1, respectively. Time of ripening accounted for (P<0.01) 67, 67 and 76% of the explained variability of histamine, tyramine, and putrescine content, respectively. Histamine, tyramine, and putrescine content in cheese (Y, mgkg−1) increased (P<0.01) linearly with increasing time of ripening (X, days): Y=0.24X−1.3 (R 2=0.28), Y=0.69X−18.9 (R 2=0.24) and Y=0.16X−1.3 (R 2=0.04), respectively. Biogenic amines (sum of BA) were produced more than three times more rapidly with increasing time of ripening in the edge part of the cheese than in the core part. Both histamine and tyramine content in cheese was negatively correlated (P<0.01) with counts of lactic acid bacteria (LAB) and counts of total anaerobes, respectively. Accordingly, only 3% of both LAB and enterococci (established tyramine producers) isolates were positive on tyrosine decarboxylase gene sequences using the PCR method.European Food Research and Technology 04/2008; 227(1):29-36. · 1.39 Impact Factor