The unique aroma of melons (Cucumis melo L., Cucurbitaceae) is composed of many volatile compounds biosynthetically derived from fatty acids, carotenoids, amino acids, and terpenes. Although amino ...acids are known precursors of aroma compounds in the plant kingdom, the initial steps in the catabolism of amino acids into aroma volatiles have received little attention. Incubation of melon fruit cubes with amino acids and α-keto acids led to the enhanced formation of aroma compounds bearing the side chain of the exogenous amino or keto acid supplied. Moreover, L-13C6phenylalanine was also incorporated into aromatic volatile compounds. Amino acid transaminase activities extracted from the flesh of mature melon fruits converted L-isoleucine, L-leucine, L-valine, L-methionine, or L-phenylalanine into their respective α-keto acids, utilizing α-ketoglutarate as the amine acceptor. Two novel genes were isolated and characterized (CmArAT1 and CmBCAT1) encoding 45.6 kDa and 42.7 kDa proteins, respectively, that displayed aromatic and branched-chain amino acid transaminase activities, respectively, when expressed in Escherichia coli. The expression of CmBCAT1 and CmArAT1 was low in vegetative tissues, but increased in flesh and rind tissues during fruit ripening. In addition, ripe fruits of climacteric aromatic cultivars generally showed high expression of CmBCAT1 and CmArAT1 in contrast to non-climacteric non-aromatic fruits. The results presented here indicate that in melon fruit tissues, the catabolism of amino acids into aroma volatiles can initiate through a transamination mechanism, rather than decarboxylation or direct aldehyde synthesis, as has been demonstrated in other plants.
L-Amino acids are the building blocks for proteins synthesized in ribosomes in all kingdoms of life, but d-amino acids (d-aa) have important non-ribosome-based functions(1). Mammals synthesize d-Ser ...and d-Asp, primarily in the central nervous system, where d-Ser is critical for neurotransmission(2). Bacteria synthesize a largely distinct set of d-aa, which become integral components of the cell wall and are also released as free d-aa(3,4). However, the impact of free microbial d-aa on host physiology at the host-microbial interface has not been explored. Here, we show that the mouse intestine is rich in free d-aa that are derived from the microbiota. Furthermore, the microbiota induces production of d-amino acid oxidase (DAO) by intestinal epithelial cells, including goblet cells, which secrete the enzyme into the lumen. Oxidative deamination of intestinal d-aa by DAO, which yields the antimicrobial product H2O2, protects the mucosal surface in the small intestine from the cholera pathogen. DAO also modifies the composition of the microbiota and is associated with microbial induction of intestinal sIgA. Collectively, these results identify d-aa and DAO as previously unrecognized mediators of microbe-host interplay and homeostasis on the epithelial surface of the small intestine.
Protein sequence matching presently fails to identify many structures that are highly similar, even when they are known to have the same function. The high packing densities in globular proteins lead ...to interdependent substitutions, which have not previously been considered for amino acid similarities. At present, sequence matching compares sequences based only upon the similarities of single amino acids, ignoring the fact that in densely packed protein, there are additional conservative substitutions representing exchanges between two interacting amino acids, such as a small‐large pair changing to a large‐small pair substitutions that are not individually so conservative. Here we show that including information for such pairs of substitutions yields improved sequence matches, and that these yield significant gains in the agreements between sequence alignments and structure matches of the same protein pair. The result shows sequence segments matched where structure segments are aligned. There are gains for all 2002 collected cases where the sequence alignments that were not previously congruent with the structure matches. Our results also demonstrate a significant gain in detecting homology for “twilight zone” protein sequences. The amino acid substitution metrics derived have many other potential applications, for annotations, protein design, mutagenesis design, and empirical potential derivation.
Amino acids and immune function Li, Peng; Yin, Yu-Long; Li, Defa ...
British journal of nutrition,
08/2007, Volume:
98, Issue:
2
Journal Article
Peer reviewed
Open access
A deficiency of dietary protein or amino acids has long been known to impair immune function and increase the susceptibility of animals and humans to infectious disease. However, only in the past 15 ...years have the underlying cellular and molecular mechanisms begun to unfold. Protein malnutrition reduces concentrations of most amino acids in plasma. Findings from recent studies indicate an important role for amino acids in immune responses by regulating: (1) the activation of T lymphocytes, B lymphocytes, natural killer cells and macrophages; (2) cellular redox state, gene expression and lymphocyte proliferation; and (3) the production of antibodies, cytokines and other cytotoxic substances. Increasing evidence shows that dietary supplementation of specific amino acids to animals and humans with malnutrition and infectious disease enhances the immune status, thereby reducing morbidity and mortality. Arginine, glutamine and cysteine precursors are the best prototypes. Because of a negative impact of imbalance and antagonism among amino acids on nutrient intake and utilisation, care should be exercised in developing effective strategies of enteral or parenteral provision for maximum health benefits. Such measures should be based on knowledge about the biochemistry and physiology of amino acids, their roles in immune responses, nutritional and pathological states of individuals and expected treatment outcomes. New knowledge about the metabolism of amino acids in leucocytes is critical for the development of effective means to prevent and treat immunodeficient diseases. These nutrients hold great promise in improving health and preventing infectious diseases in animals and humans.
Production of non‐canonical D‐amino acids (NCDAAs) in stationary phase promotes remodelling of peptidoglycan (PG), the polymer that comprises the bacterial cell wall. Impairment of NCDAAs production ...leads to excessive accumulation of PG and hypersensitivity to osmotic shock; however, the mechanistic bases for these phenotypes were not previously determined. Here, we show that incorporation of NCDAAs into PG is a critical means by which NCDAAs control PG abundance and strength. We identified and reconstituted in vitro two (of at least three) distinct processes that mediate NCDAA incorporation. Diverse bacterial phyla incorporate NCDAAs into their cell walls, either through periplasmic editing of the mature PG or via incorporation into PG precursor subunits in the cytosol. Production of NCDAAs in Vibrio cholerae requires the stress response sigma factor RpoS, suggesting that NCDAAs may aid bacteria in responding to varied environmental challenges. The widespread capacity of diverse bacteria, including non‐producers, to incorporate NCDAAs suggests that these amino acids may serve as both autocrine‐ and paracrine‐like regulators of chemical and physical properties of the cell wall in microbial communities.
Upon entry into stationary phase, various bacterial species release D‐amino acids that are incorporated into their cell walls. This study provides insight into the regulation, mechanisms and consequences of this phenomenon.
Dietary protein and amino acid requirement recommendations for normal "healthy" children and adults have varied considerably with 2007 FAO/WHO protein requirement estimates for children lower, but ...dietary essential AA requirements for adults more than doubled. Requirement estimates as presented do not account for common living conditions, which are prevalent in developing countries such as energy deficit, infection burden and added functional demands for protein and AAs. This study examined the effect of adjusting total dietary protein for quality and digestibility (PDCAAS) and of correcting current protein and AA requirements for the effect of infection and a mild energy deficit to estimate utilizable protein (total protein corrected for biological value and digestibility) and the risk/prevalence of protein inadequacy. The relationship between utilizable protein/prevalence of protein inadequacy and stunting across regions and countries was examined. Data sources (n = 116 countries) included FAO FBS (food supply), UNICEF (stunting prevalence), UNDP (GDP) and UNSTATS (IMR) and USDA nutrient tables. Statistical analyses included Pearson correlations, paired-sample/non-parametric t-tests and linear regression. Statistically significant differences were observed in risk/prevalence estimates of protein inadequacy using total protein and the current protein requirements versus utilizable protein and the adjusted protein requirements for all regions (p < 0·05). Total protein, utilizable protein, GDP per capita and total energy were each highly correlated with the prevalence of stunting. Energy, protein and utilizable protein availability were independently and negatively associated with stunting (p < 0·001), explaining 41 %, 34 % and 40 % of variation respectively. Controlling for energy, total protein was not a statistically significant factor but utilizable protein remained significant explaining~45 % of the variance (p = 0·017). Dietary utilizable protein provides a better index of population impact of risk/prevalence of protein inadequacy than crude protein intake. We conclude that the increased demand for protein due to infections and mild to moderate energy deficits, should be appropriately considered in assessing needs of populations where those conditions still prevail.
Protein intakes in India Swaminathan, Sumathi; Vaz, Mario; Kurpad, Anura V
British journal of nutrition
108 Suppl 2
Journal Article
Peer reviewed
Open access
Indian diets derive almost 60 % of their protein from cereals with relatively low digestibility and quality. There have been several surveys of diets and protein intakes in India by the National ...Nutrition Monitoring Board (NNMB) over the last 25 years, in urban and rural, as well as in slum dwellers and tribal populations. Data of disadvantaged populations from slums, tribals and sedentary rural Indian populations show that the protein intake (mainly from cereals) is about 1 gm/kg/day. However, the protein intake looks less promising in terms of the protein digestibility corrected amino acid score (PDCAAS), using lysine as the first limiting amino acid, where all populations, particularly rural and tribal, appear to have an inadequate quality to their protein intake. The protein: energy (PE) ratio is a measure of dietary quality, and has been used in the 2007 WHO/FAO/UNU report to define reference requirement values with which the adequacy of diets can be evaluated in terms of a protein quality corrected PE ratio. It is likely that about one third of this sedentary rural population is at risk of not meeting their requirements. These levels of risk of deficiency are in a population with relatively low BMI populations, whose diets are also inadequate in fruits and vegetables. Therefore, while the burden of enhancing the quality of protein intake in rural India exists, the quality of the diet, in general, represents a challenge that must be met.
D‐Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of
D‐amino acids, making it one of the most promising routes for synthesizing optically pure
L‐amino acids, including
...L‐phosphinothricin (
L‐PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate
D‐PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis (RtwDAAO), and improved its catalytic potential toward
D‐PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel‐pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M3rd‐SHVG was obtained, exhibiting a >2000‐fold increase in relative activity. The kinetic parameters showed that M3rd‐SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M3rd‐SHVG reshapes the tunnel‐pocket and corrects the direction of enzyme–substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel‐pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M3rd‐SHVG as biocatalyst, 500 mM D,
L‐PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production.
•Short-term treatment with l-Leucine or l-Isoleucine in AIN-93G diet promoted rat bladder carcinogenesis.•Supplementation with l-Leucine or l-Isoleucine triggered expression of amino acid ...transporters.•Their supplementation also up-regulated tumorigenesis-associated genes.
We investigated the underlying mechanisms of l-leucine and l-isoleucine mediated promotion of bladder carcinogenesis using an initiation-promotion model. Rats were administered N-butyl-N-(4-hydroxybutyl) nitrosamine for 4weeks and then fed AIN-93G basal diet or diet supplemented with l-leucine or l-isoleucine for 8weeks followed by the basal diet for another 8weeks. At the end of the experiment, week 20, there was a significant elevation of papillary and nodular (PN) hyperplasia multiplicity in the amino acid groups. l-Leucine and l-isoleucine transporters were up-regulated in PN hyperplasias and/or bladder tumors compared with concomitant normal-appearing bladder urothelium at weeks 12 and/or 20 in all groups. In addition, in normal-appearing bladder urothelium, significantly increased mRNA levels of y+LAT1, LAT2, LAT4, and 4F2hc were observed in the amino acid groups compared with the BBN control group at both weeks 12 and 20, and increased mRNA levels of LAT1 were observed at week 20. Furthermore, up-regulation of TNF-α, c-fos, β-catenin, p53, p21Cip1/WAF1, cdk4, cyclin D1 and caspase 3 in the amino acid groups was detected in normal-appearing bladder urothelium. Overall, our results indicate that supplementation with l-leucine or l-isoleucine enhanced growth of bladder urothelial tumors by triggering expression of amino acid transporters and tumorigenesis-associated genes.
Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. ...To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Abs
) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Abs
in healthy males (R
= 0.439, P = 0.000), females (R
= 0.245, P = 0.000), female stroke patients (R
= 0.187, P = 0.000), but not in male stroke patients (R
= 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Abs
correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Abs
, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Abs
, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Abs
were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.
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