Gut bacterial deamination of residual levodopa medication for Parkinson’s disease

Aromatic bacterial metabolites are attracting considerable attention due to their impact on gut homeostasis and host’s physiology. Clostridium sporogenes is a key contributor to the production of these bioactive metabolites in the human gut. Here, we show that C. sporogenes deaminates levodopa, the main treatment in Parkinson’s disease, and identify the aromatic aminotransferase responsible for the initiation of the deamination pathway. The deaminated metabolite from levodopa, 3-(3,4-dihydroxyphenyl)propionic acid, elicits an inhibitory effect on ileal motility in an ex vivo model. 3-(3,4-dihydroxyphenyl)propionic acid is detected in fecal samples of Parkinson’s disease patients on levodopa medication. Our data are of significant impact to the treatment of Parkinson’s disease, where constipation is reported as the most common gastrointestinal symptom. Overall, this study underpins the importance of the metabolic pathways of the gut microbiome involved in drug metabolism not only to preserve drug effectiveness, but also to avoid potential side effects of bacterial breakdown products of the unabsorbed residue of medication.

Improved l-Leucine Production in Corynebacterium glutamicum by Optimizing the Aminotransferases

The production of branched-chain amino acids (BCAAs) is still challenging, therefore we rationally engineered Corynebacterium glutamicum FA-1 to increase the l-leucine production by optimizing the aminotransferases. Based on this, we investigated the effects of the native aminotransferases, i.e., branched-chain amino acid aminotransferase (BCAT; encoded by ilvE) and aspartate aminotransferase (AspB; encoded by aspB) on l-leucine production in C. glutamicum. The strain FA-1△ilvE still exhibited significant growth without leucine addition, while FA-1△ilvE△aspB couldn’t, which indicated that AspB also contributes to L-leucine synthesis in vivo and the yield of leucine reached 20.81 ± 0.02 g/L. It is the first time that AspB has been characterized for l-leucine synthesis activity. Subsequently, the aromatic aminotransferase TyrB and the putative aspartate aminotransferases, the aspC, yhdR, ywfG gene products, were cloned, expressed and characterized for leucine synthesis activity in FA-1△ilvE△aspB. Only TyrB was able to synthesize l-leucine and the l-leucine production was 18.55 ± 0.42 g/L. The two putative branched-chain aminotransferase genes, ybgE and CaIlvE, were also cloned and expressed. Both genes products function efficiently in BCAAs biosynthesis. This is the first report of a rational modification of aminotransferase activity that improves the l-leucine production through optimizing the aminotransferases.

Identification of an Arabidopsis Aminotransferase that Facilitates Tryptophan and Auxin Homeostasis

IAA plays a critical role in regulating numerous aspects of plant growth and development. While there is much genetic support for tryptophan-dependent (Trp-D) IAA synthesis pathways, there is little genetic evidence for tryptophan-independent (Trp-I) IAA synthesis pathways. Using Arabidopsis, we identified two mutant alleles of ISS1 (Indole Severe Sensitive) that display indole-dependent IAA overproduction phenotypes including leaf epinasty and adventitious rooting. Stable isotope labeling showed that iss1, but not WT, uses primarily Trp-I IAA synthesis when grown on indole-supplemented medium. In contrast, both iss1 and WT use primarily Trp-D IAA synthesis when grown on unsupplemented medium. iss1 seedlings produce 8-fold higher levels of IAA when grown on indole and surprisingly have a 174-fold increase in Trp. These findings indicate that the iss1 mutant???s increase in Trp-I IAA synthesis is due to a loss of Trp catabolism. ISS1 was identified as At1g80360, a predicted aromatic aminotransferase, and in vitro and in vivo analysis confirmed this activity. At1g80360 was previously shown to primarily carry out the conversion of indole-3-pyruvic acid to Trp as an IAA homeostatic mechanism in young seedlings. Our results suggest that in addition to this activity, in more mature plants ISS1 has a role in Trp catabolism and possibly in the metabolism of other aromatic amino acids. We postulate that this loss of Trp catabolism impacts the use of Trp-D and/or Trp-I IAA synthesis pathways.

Genetic Characterization of the Major Lactococcal Aromatic Aminotransferase and Its Involvement in Conversion of Amino Acids to Aroma Compounds

ABSTRACT In lactococci, transamination is the first step of the enzymatic conversion of aromatic and branched-chain amino acids to aroma compounds. In previous work we purified and biochemically characterized the major aromatic aminotransferase (AraT) of a Lactococcus lactis subsp. cremoris strain. Here we characterized the corresponding gene and evaluated the role of AraT in the biosynthesis of amino acids and in the conversion of amino acids to aroma compounds. Amino acid sequence homologies with other aminotransferases showed that the enzyme belongs to a new subclass of the aminotransferase I subfamily γ; AraT is the best-characterized representative of this new aromatic-amino-acid-specific subclass. We demonstrated that AraT plays a major role in the conversion of aromatic amino acids to aroma compounds, since gene inactivation almost completely prevented the degradation of these amino acids. It is also highly involved in methionine and leucine conversion. AraT also has a major physiological role in the biosynthesis of phenylalanine and tyrosine, since gene inactivation weakly slowed down growth on medium without phenylalanine and highly affected growth on every medium without tyrosine. However, another biosynthesis aromatic aminotransferase is induced in the absence of phenylalanine in the culture medium.

Purification and properties of aromatic amino acid aminotransferases from Azospirillum brasilense UAP 14 strain

The purification and characterization of AAT1, one of two aromatic amino acid aminotransferase (EC 2.6.1.57) in Azospirillum brasilense, is described. Purified AAT1 had a subunit mass of 33 kDa and a nondenatured molecular mass of 66 kDa, suggesting a dimeric structure. Other properties include a pI of 5.04, an optimum temperature of 45 °C, and optimum pH of 8.5. AAT1 utilized all aromatic amino acids, the L-tryptophan derivatives such as L-5-methyl tryptophan and L-flourtryptophan, and L-histidine. The apparent Km values for L-tyrosine, L-phenylalanine, and L-tryptophan were 0.19, 0.43, and 1.05 mM, respectively. The enzyme was competive inhibited by indole-3-pyruvic acid with a Ki of 0.17 mM.Key words: aromatic aminotransferase, Azospirillum brasilense, indole acetic acid production.