Aromatic Amino Acid Aminotransferase Research Articles

Uncovering the specificity and predictability of tryptophan metabolism in lactic acid bacteria with genomics and metabolomics.

Tryptophan is metabolized by microorganisms into various indole derivatives that have been proven to alleviate diseases and promote human health. Lactic acid bacteria (LAB) are a broad microbial concept, some of which have been developed as probiotics. However, the capacity of most LAB to metabolize tryptophan is unknown. In this study, the aim is to reveal the rule of tryptophan metabolism in LAB by multi-omics. The findings showed that LAB were rich in genes for tryptophan catabolism and that multiple genes were shared among LAB species. Although the number of their homologous sequences was different, they could still form the same metabolic enzyme system. The metabolomic analysis revealed that LAB were capable of producing a variety of metabolites. Strains belonging to the same species can produce the same metabolites and have similar yields. A few strains showed strain-specificity in the production of indole-3-lactic acid (ILA), indole-3-acetic acid, and 3-indolealdehyde (IAld). In the genotype-phenotype association analysis, the metabolites of LAB were found to be highly consistent with the outcomes of gene prediction, particularly ILA, indole-3-propionic acid, and indole-3-pyruvic acid. The overall prediction accuracy was more than 87% on average, which indicated the predictability of tryptophan metabolites of LAB. Additionally, genes influenced the concentration of metabolites. The levels of ILA and IAld were significantly correlated with the numbers of aromatic amino acid aminotransferase and amidase, respectively. The unique indolelactate dehydrogenase in Ligilactobacillus salivarius was the primary factor contributing to its large production of ILA. In summary, we demonstrated the gene distribution and production level of tryptophan metabolism in LAB and explored the correlation between genes and phenotypes. The predictability and specificity of the tryptophan metabolites in LAB were proven. These results provide a novel genomic method for the discovery of LAB with tryptophan metabolism potential and offer experimental data for probiotics that produce specific tryptophan metabolites.

Effect of the biosynthesis of the volatile compound phenylacetaldehyde on chloroplast modifications in tea (Camellia sinensis) plants.

Plant volatile compounds have important physiological and ecological functions. Phenylacetaldehyde (PAld), a volatile phenylpropanoid/benzenoid, accumulates in the leaves of tea (Camellia sinensis) plants grown under continuous shading. This study was conducted to determine whether PAld production is correlated with light and to elucidate the physiological functions of PAld in tea plants. Specifically, the upstream mechanism modulating PAld biosynthesis in tea plants under different light conditions as well as the effects of PAld on chloroplast/chlorophyll were investigated. The biosynthesis of PAld was inhibited under light, whereas it was induced in darkness. The structural gene encoding aromatic amino acid aminotransferase 1 (CsAAAT1) was expressed at a high level in darkness, consistent with its importance for PAld accumulation. Additionally, the results of a transcriptional activation assay and an electrophoretic mobility shift assay indicated CsAAAT1 expression was slightly activated by phytochrome-interacting factor 3-2 (CsPIF3-2), which is a light-responsive transcription factor. Furthermore, PAld might promote the excitation of chlorophyll in dark-treated chloroplasts and mediate electron energy transfer in cells. However, the accumulated PAld can degrade chloroplasts and chlorophyll, with potentially detrimental effects on photosynthesis. Moreover, PAld biosynthesis is inhibited in tea leaves by red and blue light, thereby decreasing the adverse effects of PAld on chloroplasts during daytime. In conclusion, the regulated biosynthesis of PAld in tea plants under light and in darkness leads to chloroplast modifications. The results of this study have expanded our understanding of the biosynthesis and functions of volatile phenylpropanoids/benzenoids in tea leaves.

Microbial diversity and functional genes of red vinasse acid based on metagenome analysis.

Red vinasse acid has a distinct flavor and a vivid red color that are directly tied to the intricate metabolic activities of microorganisms that produce it. In this study, metagenomic technology was used to mine its functional genes and examine the microbial diversity of red vinasse acid. The findings revealed the identification of 2,609 species, 782 genera, and 63 phyla of microorganisms, and the dominant genus was Lactobacillus. Amino acid metabolism and carbohydrate metabolism were significant activities among the 16,093 and 49,652 genes that were annotated in the evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. In gluconeogenesis, red vinasse acid encodes 194 genes controlling the transporter protein systems of different sugars and has key enzyme genes that catalyze the conversion of intracellular sugars into glycolytic intermediates. In amino acid flavor formation, red vinasse acid contains 32 control genes for branched-chain aminotransferase (BCAT), 27 control genes for aromatic-amino-acid transaminase (ArAT), 60 control genes for keto acid invertase, 123 control genes for alcohol/aldehyde dehydrogenase, and 27 control genes for acetyl esterase, which have the basis for the formation of strong flavor substances from amino acids.

Loss of YggS (COG0325) impacts aspartate metabolism in Salmonella enterica.

YggS is a pyridoxal 5'-phosphate (PLP)-binding protein of the conserved COG0325 family. Despite a connection with vitamin B6 homeostasis in many species, neither a precise biochemical activity nor the molecular mechanism of how YggS contributes to cellular function has been described. In a transposon mutagenesis screen, we found that insertions in aspC (encoding a PLP-dependent aspartate aminotransferase, EC 2.6.1.1) in a Salmonella enterica strain lacking yggS caused a synthetic growth defect, which could be rescued by the addition of exogenous aspartate. Characterization of spontaneous suppressors which improved the growth of the yggS aspC double mutant suggested that this synthetic aspartate limitation was dependent on TyrB, a PLP-dependent aromatic amino acid aminotransferase (EC 2.6.1.57). Genetic and biochemical data were consistent with the hypothesis that TyrB activity was inhibited by accumulated pyridoxine 5'-phosphate and α-keto acids caused by a yggS mutation. This study provides data consistent with a working model implicating YggS in modulating concentrations of B6 vitamers via transamination.

Structural Evidence of Active Site Adaptability towards Different Sized Substrates of Aromatic Amino Acid Aminotransferase from Psychrobacter Sp. B6.

Aromatic amino acid aminotransferases present a special potential in the production of drugs and synthons, thanks to their ability to accommodate a wider range of substrates in their active site, in contrast to aliphatic amino acid aminotransferases. The mechanism of active site adjustment toward substrates of psychrophilic aromatic amino acid aminotransferase (PsyArAT) from Psychrobacter sp. B6 is discussed based on crystal structures of complexes with four hydroxy-analogs of substrates: phenylalanine, tyrosine, tryptophan and aspartic acid. These competitive inhibitors are bound in the active center of PsyArAT but do not undergo transamination reaction, which makes them an outstanding tool for examination of the enzyme catalytic center. The use of hydroxy-acids enabled insight into substrate binding by native PsyArAT, without mutating the catalytic lysine and modifying cofactor interactions. Thus, the binding mode of substrates and the resulting analysis of the volume of the catalytic site is close to a native condition. Observation of these inhibitors’ binding allows for explanation of the enzyme’s adaptability to process various sizes of substrates and to gain knowledge about its potential biotechnological application. Depending on the character and size of the used inhibitors, the enzyme crystallized in different space groups and showed conformational changes of the active site upon ligand binding.

Lemon Exosome-like Nanoparticles-Manipulated Probiotics Protect Mice from C.d iff Infection.

SummaryClostridioides difficile (C. diff) is the leading cause of antibiotic-associated colitis. Here, we report that lemon exosome-like nanoparticles (LELNs) manipulated probiotics to inhibit C. diff infection (CDI). LELN-manipulated Lactobacillus rhamnosus GG (LGG) and Streptococcus thermophilus ST-21 (STH) (LELN-LS) decrease CDI mortality via an LELN-mediated increase in bile resistance and gut survivability. LELN-LS treatment increases the AhR ligands indole-3-lactic acid (I3LA) and indole-3-carboxaldehyde (I3Ald), leading to induction of IL-22, and increases lactic acid leading to a decrease of C. diff fecal shedding by inhibiting C. diff growth and indole biosynthesis. A synergistic effect between STH and LGG was identified. The STH metabolites inhibit gluconeogenesis of LGG and allow fructose-1,6-bisphosphate (FBP) to accumulate in LGG; accumulated FBP then activates lactate dehydrogenase of LGG (LGG-LDH) and enhances production of lactic acid and the AhR ligand. Our findings provide a new strategy for CDI prevention and treatment with a new type of prebiotics.

In vivo cascade catalysis of aromatic amino acids to the respective mandelic acids using recombinant E. coli cells expressing hydroxymandelate synthase (HMS) from Amycolatopsis mediterranei

Mandelic acids are valuable products which are used in a broad field of applications. The enzyme hydroxymandelate synthase (HMS) is a non-heme iron dioxygenase which converts para-hydroxyphenylpyruvate and other 3-aryl pyruvates by decarboxylation to the corresponding mandelates. In the present work, the gene hms encoding the hydroxymandelate synthase from Amycolatopsis mediterranei was cloned and overexpressed in Escherichia coli BL21(DE3) for in vivo cascade catalysis taking advantage of resident aromatic amino acid transaminases. The resulting recombinant cells were used for whole cell biotransformations. We successfully accomplished the production of para-hydroxymandelate exclusively by using the aromatic amino acid l-tyrosine in biotransformation. Furthermore, by utilizing different phenylalanine derivatives (including chloro-, fluoro- and hydroxylated amino acids), the corresponding S-mandelic acids were obtained with high conversion (21–87 %) and high ee (38–97%). This process is an alternative and attractive way to get access to a variety of mandelic acids.

Efficient Synthesis of Hydroxytyrosol from l-3,4-Dihydroxyphenylalanine Using Engineered Escherichia coli Whole Cells.

Hydroxytyrosol is a high-value-added compound with a variety of biological and pharmacological activities. In this study, a whole-cell catalytic method for the synthesis of hydroxytyrosol was developed: aromatic amino acid aminotransferase (TyrB), l-glutamate dehydrogenase (GDH), α-keto acid decarboxylase (PmKDC), and aldehyde reductase (YahK) were co-expressed in Escherichia coli to catalyze the synthesis of hydroxytyrosol from l-3,4-dihydroxyphenylalanine (l-DOPA). The plasmids with different copy numbers were used to balance the expression of the four enzymes, and the most appropriate strain (pRSF- yahK- tyrB and pCDF- gdh- Pmkdc) was identified. After determination of the optimum temperature (35 °C) and pH (7.5) for whole-cell catalysis, the yield of hydroxytyrosol reached 36.33 mM (5.59 g/L) and the space-time yield reached 0.70 g L-1 h-1.

Biochemical Characterization of Aspergillus fumigatus AroH, a Putative Aromatic Amino Acid Aminotransferase.

The rise in the frequency of nosocomial infections is becoming a major problem for public health, in particular in immunocompromised patients. Aspergillus fumigatus is an opportunistic fungus normally present in the environment directly responsible for lethal invasive infections. Recent results suggest that the metabolic pathways related to amino acid metabolism can regulate the fungus-host interaction and that an important role is played by enzymes involved in the catabolism of L-tryptophan. In particular, in A. fumigatus L-tryptophan regulates Aro genes. Among them, AroH encodes a putative pyridoxal 5'-phosphate-dependent aminotransferase. Here we analyzed the biochemical features of recombinant purified AroH by spectroscopic and kinetic analyses corroborated by in silico studies. We found that the protein is dimeric and tightly binds the coenzyme forming a deprotonated internal aldimine in equilibrium with a protonated ketoenamine form. By setting up a new rapid assay method, we measured the kinetic parameters for the overall transamination of substrates and we demonstrated that AroH behaves as an aromatic amino acid aminotransferase, but also accepts L-kynurenine and α-aminoadipate as amino donors. Interestingly, computational approaches showed that the predicted overall fold and active site topology of the protein are similar to those of its yeast ortholog, albeit with some differences in the regions at the entrance of the active site, which could possibly influence substrate specificity. Should targeting fungal metabolic adaptation be of therapeutic value, the results of the present study may pave the way to the design of specific AroH modulators as potential novel agents at the host/fungus interface.

Increasing postharvest high-temperatures lead to increased volatile phenylpropanoids/benzenoids accumulation in cut rose (Rosa hybrida) flowers

Fragrance is an important quality index of horticultural flowers. Floral volatile formation in flowers during plant growth has been widely studied, but less is known about floral volatile formation in cut flowers and its responses to postharvest conditions. In this study, cut rose (Rosa hybrida cv. Tineke) flowers subjected to 5, 15 and 30 °C for 36 h showed increased concentrations of volatile phenylpropanoids/benzenoids (VPBs) including 2-phenylethanol (2PE), phenylacetaldehyde, benzyl alcohol, benzaldehyde, and phenethyl acetate, but a reduced 3,5-dimethoxytoluene concentration, as temperatures increased. l-[2H8]Phenylalanine (Phe) tracing in vivo suggested that phenylpyruvic acid (PPA) was involved in the increase in 2PE in response to increasing temperature. Genes for two aromatic amino acid aminotransferases (AAATs) were isolated and functionally characterized. Transient expression analyses in Nicotiana benthamiana plants provided in vivo evidence that RhAAAT2 was able to convert l-Phe into PPA, and that it was localized in the cytoplasm. These results advance our understanding of floral aroma formation in flowers after harvest.

An alternative pathway for the formation of aromatic aroma compounds derived from l-phenylalanine via phenylpyruvic acid in tea (Camellia sinensis (L.) O. Kuntze) leaves

Aromatic aroma compounds contribute to flavor of tea (Camellia sinensis (L.) O. Kuntze) and they are mostly derived from l-phenylalanine via trans-cinnamic acid or directly from l-phenylalanine. The objective of this study was to investigate whether an alternative pathway derived from l-phenylalanine via phenylpyruvic acid is involved in formation of aroma compounds in tea. Enzyme reaction with phenylpyruvic acid showed that benzaldehyde, benzyl alcohol, and methyl benzoate were derived from phenylpyruvic acid in tea leaves. Feeding experiments using [2H8]l-phenylalanine indicated that phenylpyruvic acid was derived from l-phenylalanine in a reaction catalyzed by aromatic amino acid aminotransferases (AAATs). CsAAAT1 showed higher catalytic efficiency towards l-phenylalanine (p ≤ 0.001) while CsAAAT2 showed higher catalytic efficiency towards l-tyrosine (p ≤ 0.001). Both CsAAATs were localized in the cytoplasm of leaf cells. In conclusion, an alternative pathway for the formation of aromatic aroma compounds derived from l-phenylalanine via phenylpyruvic acid occurred in tea leaves.

Physicochemical, taste, and functional changes during the enhanced fermentation of low-salt Sufu paste, a Chinese fermented soybean food

ABSTRACTIn this study, the physicochemical, taste, and functional changes in low-salt Sufu paste were investigated during fermentation as enhanced by a mixed starter (Pichia fermentans, Kodamaea ohmeri, and Lactococcus lactis subsp. lactis) and α-ketoglutarate. The total free amino acids increased from 0.02 mg/g dry matter to 12.63 mg/g dry matter, and monosodium glutamate-like was the major free amino acid group (3.89 mg/g dry matter), followed by bitter according to the taste characteristics. Functional components such as γ-aminobutyric acid, riboflavin (VB2), and puerarin significantly increased to 30.96, 4.97, and 12.03 mg/g dry matter, respectively, at the end of post-ripening (p < 0.05). Protease, lipase, peptidase, α-amylase, aromatic amino acid aminotransferase, and branched-chain amino acid aminotransferase activities increased and were significantly correlated (p < 0.05) with most physicochemical and functional components, indicating that enzymes may play an important role in the fermentation process of Sufu paste. Additionally, the color of the Sufu paste changed from pale yellow to yellowish brown, whereas the Sufu paste fermented without the mixed starter and α-ketoglutarate changed to gray, the unique color of gray Sufu. The preliminary results indicated that the mixed starter and α-ketoglutarate are beneficial for taste quality and color and that fermentation may be an effective method to enhance the functional components in Sufu paste. This information would be useful for future improvements in the manufacturing process and quality of Sufu paste.

Aromatic amino acid aminotransferases in plants

Aromatic amino acid aminotransferases (AAA-ATs) catalyze the reversible transamination reactions of proteinogenic and non-proteinogenic aromatic amino acids to corresponding keto acids and vice versa. The products of plant AAA-ATs serve as key precursors of many primary and secondary metabolites that are crucial for both plant and human metabolism and physiology. In most microbes, l-tyrosine (Tyr) and l-phenylalanine (Phe) aminotransferases (Tyr and Phe-ATs) catalyze the final steps of Phe and Tyr biosynthesis. On the other hand, plants use different pathways to synthesize Tyr and Phe via arogenate, in which prephenate-specific aminotransferases (PPA-ATs) catalyze the committed step in the plastids. Plant Tyr and Phe-ATs, unlike microbial counterparts, often prefer the reverse reactions and metabolize Tyr and Phe to their respective aromatic keto acids, which serve as precursors of various plant natural products (e.g. benzenoid volatiles, tocochromanols, plastoquinone, and tropane and benzylisoquinoline alkaloids). Unlike plastidic PPA-ATs, plant Tyr/Phe-ATs are localized outside of the plastids, have broad substrate specificity, and interlink Tyr and Phe metabolism. l-Tryptophan (Trp) aminotransferases (Trp-ATs) are involved in biosynthesis of the plant hormone auxin. Although significant advancement has been made on biochemical, molecular, and genetic characterizations of plant AAA-ATs, there are still many critical knowledge gaps, which are highlighted in the current review.

Engineering Eschericha coli for Enhanced Tyrosol Production.

Tyrosol is a phenolic compound found in olive oil and wines. The health benefits of tyrosol have attracted considerable attention. Because the tyrosol extraction from plants poses a major obstacle, biosynthesizing this compound using microbial hosts is of interest. In this study, the phenylpyruvate decarboxylase gene ARO10 and the aromatic amino acid aminotransferase gene ARO8 were introduced into Escherichia coli to generate two recombinant tyrosol producers. Deleting the prephenate dehydratase gene pheA and the phenylacetaldehyde dehydrogenase gene feaB improved the tyrosol production. Under the optimal fermentation conditions, a recombinant strain overexpressing ARO10 gene produced 4.15 mM tyrosol from 1% (w/v) glucose during a 48 h shake flask cultivation. Furthermore, when tyrosine was used as the substrate, the recombinant strain co-overexpressing ARO8 and ARO10 genes displayed a higher tyrosol yield, in which 8.71 mM tyrosol was produced from 10 mM tyrosine. This investigation suggests that microbial tyrosol production has application potential.

Conversion of cysteine to 3-mercaptopyruvic acid by bacterial aminotransferases.

3-Mercaptopyruvate (3MPy), a structural analog of 3-mercaptopropionic acid, is a precursor compound for biosynthesis of polythioesters in bacteria. The cost-effectiveness and sustainability of the whole process could be greatly improved by using the cysteine degradation pathway for an intracellular supply of 3MPy. Transamination of cysteine to its corresponding α-keto acid 3MPy is catalyzed by cysteine aminotransferases (CAT). However, CAT activity has so far not been described for bacterial aminotransferases (AT), and it was unknown whether they can be applied for the conversion of cysteine to 3MPy. In this study, we selected eight bacterial aminotransferases based on sequence homology to CAT of Rattus norvegicus (Got1). The aminotransferases included four aspartate aminotransferases (AATs) and four aromatic amino acid aminotransferases (ArATs) from Advenella mimigardefordensis DPN7, Escherichia coli MG1655, Shimwellia blattae ATCC 33430, Ralstonia eutropha H16 and Paracoccus denitrificans PD1222. For a more detailed characterization, all selected AAT or ArAT encoding genes were heterologously expressed in E. coli and purified. CAT activity was detected for all aminotransferases when a novel continuous coupled enzyme assay was applied. Kinetic studies revealed the highest catalytic efficiency of 5.1mM/s for AAT from A. mimigardefordensis. Formation of 3MPy from cysteine could additionally be verified by an optimized approach using derivatization of 3MPy with the Girard T reagent and liquid chromatography-mass spectrometry analyses.

Crystal structures of Mycobacterium tuberculosis HspAT and ArAT reveal structural basis of their distinct substrate specificities.

Aminotransferases of subfamily Iβ, which include histidinol phosphate aminotransferases (HspATs) and aromatic amino acid aminotransferases (ArATs), are structurally similar but possess distinct substrate specificities. This study, encompassing structural and biochemical characterisation of HspAT and ArAT from Mycobacterium tuberculosis demonstrates that the residues lining the substrate binding pocket and N-terminal lid are the primary determinants of their substrate specificities. In mHspAT, hydrophilic residues in the substrate binding pocket and N-terminal lid allow the entry and binding of its preferential substrate, Hsp. On the other hand, the hydrophobic nature of both the substrate binding pocket and the N-terminal lid of mArAT is responsible for the discrimination of a polar substrate such as Hsp, while facilitating the binding of Phe and other aromatic residues such as Tyr and Trp. In addition, the present study delineates the ligand induced conformational rearrangements, providing insights into the plasticity of aminotransferases. Furthermore, the study also demonstrates that the adventitiously bound ligand 2-(N-morpholino)ethanesulfonic acid (MES) is indeed a specific inhibitor of HspAT. These results suggest that previously untapped morpholine-ring scaffold compounds could be explored for the design of new anti-TB agents.

Micromolar colorimetric detection of 2-hydroxy ketones with the water-soluble tetrazolium WST-1

The archetypal triphenyl tetrazolium chloride (tetrazolium red) has been used in spectrophotometric microplate assays for 2-hydroxy ketones and, by extension, for the activity of enzymes including aminotransferase, transketolase, and pyruvate decarboxylase. Better sensitivity, dynamic range, and reproducibility of this method may be achieved by (i) in situ solubilization of the colored formazan with Cremophor EL and (ii) use of the newer-generation tetrazolium salts tetrazolium violet, iodonitrotetrazolium, or WST-1 (water-soluble tetrazolium). Two to 125 μM hydroxy pyruvate, dihydroxy acetone, and indole pyruvate produced by serine:pyruvate, β-alanine (ω-amino acid):pyruvate, and aromatic amino acid aminotransferase, respectively, could be detected with WST-1.

Ethylene and 1-MCP regulate major volatile biosynthetic pathways in apple fruit

The effects of ethylene and 1-methylcyclopropene (1-MCP) on apple fruit volatile biosynthesis and gene expression were investigated. Statistical analysis identified 17 genes that changed significantly in response to ethylene and 1-MCP treatments. Genes encoding branched-chain amino acid aminotransferase (BCAT), aromatic amino acid aminotransferase (ArAT) and amino acid decarboxylases (AADC) were up-regulated during ripening and further enhanced by ethylene treatment. Genes related to fatty acid synthesis and metabolism, including acyl-carrier-proteins (ACPs), malonyl-CoA:ACP transacylase (MCAT), acyl-ACP-desaturase (ACPD), lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC2), β-oxidation, acyl-CoA synthetase (ACS), enoyl-CoA hydratase (ECHD), acyl-CoA dehydrogenase (ACAD), and alcohol acyltransferases (AATs) also increased during ripening and in response to ethylene treatment. Allene oxide synthase (AOS), alcohol dehydrogenase 1 (ADH1), 3-ketoacyl-CoA thiolase and branched-chain amino acid aminotransferase 2 (BCAT2) decreased in ethylene-treated fruit. Treatment with 1-MCP and ethylene generally produced opposite effects on related genes, which provides evidence that regulation of these genes is ethylene dependent.

Identification and molecular characterization of the homogentisate pathway responsible for pyomelanin production, the major melanin constituents in Aeromonas media WS.

The pigmentation of many Aeromonas species has been thought to be due to the production of a L-DOPA (L-3,4-dihydroxyphenylalanine) based melanin. However, in this study we found that although L-DOPA synthesis occurs in the high-melanin-yielding Aeromonas media strain WS, it plays a minor, if any, role in pigmentation. Instead, the pigmentation of A. media strain WS is due to the production of pyomelanin through HGA (homogentisate). Gene products of phhA (encodes phenylalanine hydroxylase), tyrB and aspC (both encode aromatic amino acid aminotransferase), and hppD (encodes 4-hydroxyphenylpyruvate dioxygenase) constitute a linear pathway of converting phenylalanine to HGA and disruption of any one of these genes impairs or blocks pigmentation of A. media strain WS. This HGA biosynthesis pathway is widely distributed in Aeromonas, but HGA is only detectable in the cultures of pigmented Aeromonas species. Heterologous expression of HppD from both pigmented and non-pigmented Aeromonas species in E. coli leads to the production of pyomelanin and thus pigmentation, suggesting that most Aeromonas species have the critical enzymes to produce pyomelanin through HGA. Taken together, we have identified a widely conserved biosynthesis pathway of HGA based pyomelanin in Aeromonas that may be responsible for pigmentation of many Aeromonas species.

Crystal structure and enzymatic properties of a broad substrate-specificity psychrophilic aminotransferase from the Antarctic soil bacteriumPsychrobactersp. B6

Aminotransferases (ATs) are enzymes that are commonly used in the chemical and pharmaceutical industries for the synthesis of natural and non-natural amino acids by transamination reactions. Currently, the easily accessible enzymes from mesophilic organisms are most commonly used; however, for economical and ecological reasons the utilization of aminotransferases from psychrophiles would be more advantageous, as their optimum reaction temperature is usually significantly lower than for the mesophilic ATs. Here, gene isolation, protein expression, purification, enzymatic properties and structural studies are reported for the cold-active aromatic amino-acid aminotransferase (PsyArAT) from Psychrobacter sp. B6, a psychrotrophic, Gram-negative strain from Antarctic soil. Preliminary computational analysis indicated dual functionality of the enzyme through the ability to utilize both aromatic amino acids and aspartate as substrates. This postulation was confirmed by enzymatic activity tests, which showed that it belonged to the class EC 2.6.1.57. The first crystal structures of a psychrophilic aromatic amino-acid aminotransferase have been determined at resolutions of 2.19 Å for the native enzyme (PsyArAT) and 2.76 Å for its complex with aspartic acid (PsyArAT/D). Both types of crystals grew in the monoclinic space group P21 under slightly different crystallization conditions. The PsyArAT crystals contained a dimer (90 kDa) in the asymmetric unit, which corresponds to the active form of this enzyme, whereas the crystals of the PsyArAT/D complex included four dimers showing different stages of the transamination reaction.