Decoding the Complex Crossroad of Tryptophan Metabolic Pathways

Among the 20 amino acids needed for protein synthesis, Tryptophan (Trp) is an aromatic amino acid fundamental not only for the synthesis of the major components of living cells (namely, the proteins), but also for the maintenance of cellular homeostasis [...]

Clustering of Aromatic Amino Acid Residues around Methionine in Proteins

Short-range, non-covalent interactions between amino acid residues determine protein structures and contribute to protein functions in diverse ways. The interactions of the thioether of methionine with the aromatic rings of tyrosine, tryptophan, and/or phenylalanine has long been discussed and such interactions are favorable on the order of 1–3 kcal mol−1. Here, we carry out a new bioinformatics survey of known protein structures where we assay the propensity of three aromatic residues to localize around the [-CH2-S-CH3] of methionine. We term these groups “3-bridge clusters”. A dataset consisting of 33,819 proteins with less than 90% sequence identity was analyzed and such clusters were found in 4093 structures (or 12% of the non-redundant dataset). All sub-classes of enzymes were represented. A 3D coordinate analysis shows that most aromatic groups localize near the CH2 and CH3 of methionine. Quantum chemical calculations support that the 3-bridge clusters involve a network of interactions that involve the Met-S, Met-CH2, Met-CH3, and the π systems of nearby aromatic amino acid residues. Selected examples of proposed functions of 3-bridge clusters are discussed.

Thermococcus sp. KS-1 PPIase as a fusion partner improving soluble production of aromatic amino acid decarboxylase

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) catalyzes the racemization reaction of proline residues on a polypeptide chain. This enzyme is also known to function as a molecular chaperon to stabilize protein conformation during the folding process. In this study, we noted FK506 binding protein (FKBP)-type PPIase from a hyperthemophilic archaeon Thermococcus sp. strain KS-1 (PPIase KS−1) to improve the solubility of Pseudomonas putida aromatic amino acid decarboxylase (AADC) that is an indispensable enzyme for fermentative production of plant isoquinoline alkaloids. AADC fused N-terminally with the PPIase KS−1 (PPIase KS−1-AADC), which was synthesized utilizing Escherichia coli host, showed improved solubility and, consequently, the cell-free extract from the recombinant strain exhibited 2.6- to 3.4-fold elevated AADC activity than that from the control strain that expressed the AADC gene without PPIase KS−1. On the other hand, its thermostability was slightly decreased by fusing PPIase KS−1. The recombinant E. coli cells expressing the PPIase KS−1-AADC gene produced dopamine and phenylethylamine from L-dopa and phenylalanine by two- and threefold faster, respectively, as compared with the control strain. We further demonstrated that the efficacy of PPIase KS−1-AADC in solubility and activity enhancement was a little but obviously higher than that of AADC fused N-terminally with NusA protein, which has been assumed to be the most effective protein solubilizer. These results suggest that PPIase KS−1 can be used as one of the best choices for producing heterologous proteins as active forms in E. coli.

Staphylococcus aureus Transcriptome Data and Metabolic Modelling Investigate the Interplay of Ser/Thr Kinase PknB, Its Phosphatase Stp, the glmR/yvcK Regulon and the cdaA Operon for Metabolic Adaptation

Serine/threonine kinase PknB and its corresponding phosphatase Stp are important regulators of many cell functions in the pathogen S. aureus. Genome-scale gene expression data of S. aureus strain NewHG (sigB+) elucidated their effect on physiological functions. Moreover, metabolic modelling from these data inferred metabolic adaptations. We compared wild-type to deletion strains lacking pknB, stp or both. Ser/Thr phosphorylation of target proteins by PknB switched amino acid catabolism off and gluconeogenesis on to provide the cell with sufficient components. We revealed a significant impact of PknB and Stp on peptidoglycan, nucleotide and aromatic amino acid synthesis, as well as catabolism involving aspartate transaminase. Moreover, pyrimidine synthesis was dramatically impaired by stp deletion but only slightly by functional loss of PknB. In double knockouts, higher activity concerned genes involved in peptidoglycan, purine and aromatic amino acid synthesis from glucose but lower activity of pyrimidine synthesis from glucose compared to the wild type. A second transcriptome dataset from S. aureus NCTC 8325 (sigB-) validated the predictions. For this metabolic adaptation, PknB was found to interact with CdaA and the yvcK/glmR regulon. The involved GlmR structure and the GlmS riboswitch were modelled. Furthermore, PknB phosphorylation lowered the expression of many virulence factors, and the study shed light on S. aureus infection processes.

L. rhamnosus improves the immune response and tryptophan catabolism in laying hen pullets

In mammals, early-life probiotic supplementation is a promising tool for preventing unfavourable, gut microbiome-related behavioural, immunological, and aromatic amino acid alterations later in life. In laying hens, feather-pecking behaviour is proposed to be a consequence of gut-brain axis dysregulation. Lactobacillus rhamnosus decreases stress-induced severe feather pecking in adult hens, but whether its effect in pullets is more robust is unknown. Consequently, we investigated whether early-life, oral supplementation with a single Lactobacillus rhamnosus strain can prevent stress-induced feather-pecking behaviour in chickens. To this end, we monitored both the short- and long-term effects of the probiotic supplement on behaviour and related physiological parameters. We hypothesized that L. rhamnosus would reduce pecking behaviour by modulating the biological pathways associated with this detrimental behaviour, namely aromatic amino acid turnover linked to neurotransmitter production and stress-related immune responses. We report that stress decreased the proportion of cytotoxic T cells in the tonsils (P = 0.047). Counteracting this T cell depression, birds receiving the L. rhamnosus supplementation significantly increased all T lymphocyte subset proportions (P < 0.05). Both phenotypic and genotypic feather peckers had lower plasma tryptophan concentrations compared to their non-pecking counterparts. The probiotic supplement caused a short-term increase in plasma tryptophan (P < 0.001) and the TRP:(PHE + TYR) ratio (P < 0.001). The administration of stressors did not significantly increase feather pecking in pullets, an observation consistent with the age-dependent onset of pecking behaviour. Despite minimal changes to behaviour, our data demonstrate the impact of L. rhamnosus supplementation on the immune system and the turnover of the serotonin precursor tryptophan. Our findings indicate that L. rhamnosus exerts a transient, beneficial effect on the immune response and tryptophan catabolism in pullets.

A low aromatic amino-acid diet improves renal function and prevent kidney fibrosis in mice with chronic kidney disease

Despite decades of use of low protein diets (LPD) in the management of chronic kidney disease (CKD), their mechanisms of action are unclear. A reduced production of uremic toxins could contribute to the benefits of LPDs. Aromatic amino-acids (AA) are precursors of major uremic toxins such as p-cresyl sulfate (PCS) and indoxyl sulfate (IS). We hypothesize that a low aromatic amino acid diet (LA-AAD, namely a low intake of tyrosine, tryptophan and phenylalanine) while being normoproteic, could be as effective as a LPD, through the decreased production of uremic toxins. Kidney failure was chemically induced in mice with a diet containing 0.25% (w/w) of adenine. Mice received three different diets for six weeks: normoproteic diet (NPD: 14.7% proteins, aromatic AAs 0.019%), LPD (5% proteins, aromatic AAs 0.007%) and LA-AAD (14% proteins, aromatic AAs 0.007%). Both LPD and LA-AAD significantly reduced proteinuria, kidney fibrosis and inflammation. While LPD only slightly decreased plasma free PCS and free IS compared to NPD; free fractions of both compounds were significantly decreased by LA-AAD. These results suggest that a LA-AAD confers similar benefits of a LPD in delaying the progression of CKD through a reduction in some key uremic toxins production (such as PCS and IS), with a lower risk of malnutrition.