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.

Crystal structure of the aromatic-amino-acid aminotransferase fromStreptococcus mutans

Streptococcus mutans, a facultatively aerobic and Gram-positive bacterium, is the primary causative agent of dental caries and contributes to the multispecies biofilm known as dental plaque. In this study, the aromatic-amino-acid aminotransferase fromStreptococcus mutans(SmAroAT) was recombinantly expressed inEscherichia coli. An effective purification protocol was established. The recombinant protein was crystallized using the hanging-drop vapor-diffusion method with PEG 3350 as the primary precipitant. The crystal structure ofSmAroAT was solved at 2.2 Å resolution by the molecular-replacement method. Structural analysis indicated that the proteins of the aromatic-amino-acid aminotransferase family have conserved structural elements that might play a role in substrate binding. These results may help in obtaining a better understanding of the catabolism and biosynthesis of aromatic amino acids.

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) fromPsychrobactersp. 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 groupP21under slightly different crystallization conditions. ThePsyArAT crystals contained a dimer (90 kDa) in the asymmetric unit, which corresponds to the active form of this enzyme, whereas the crystals of thePsyArAT/D complex included four dimers showing different stages of the transamination reaction.

Histidine Degradation via an Aminotransferase Increases the Nutritional Flexibility of Candida glabrata

ABSTRACTThe ability to acquire nutrients during infections is an important attribute in microbial pathogenesis. Amino acids are a valuable source of nitrogen if they can be degraded by the infecting organism. In this work, we analyzed histidine utilization in the fungal pathogen of humansCandida glabrata. Hemiascomycete fungi, likeC. glabrataorSaccharomyces cerevisiae, possess no gene coding for a histidine ammonia-lyase, which catalyzes the first step of a major histidine degradation pathway in most other organisms. We show thatC. glabratainstead initializes histidine degradation via the aromatic amino acid aminotransferase Aro8. AlthoughARO8is also present inS. cerevisiaeand is induced by extracellular histidine, the yeast cannot use histidine as its sole nitrogen source, possibly due to growth inhibition by a downstream degradation product. Furthermore,C. glabratarelies only on Aro8 for phenylalanine and tryptophan utilization, sinceARO8, but not its homologueARO9, was transcriptionally activated in the presence of these amino acids. Accordingly, anARO9deletion had no effect on growth with aromatic amino acids. In contrast, inS. cerevisiae,ARO9is strongly induced by tryptophan and is known to support growth on aromatic amino acids. Differences in the genomic structure of theARO9gene betweenC. glabrataandS. cerevisiaeindicate a possible disruption in the regulatory upstream region. Thus, we show that, in contrast toS. cerevisiae,C. glabratahas adapted to use histidine as a sole source of nitrogen and that the aromatic amino acid aminotransferase Aro8, but not Aro9, is the enzyme required for this process.