scholarly journals Simultaneous quantification of depolymerization and mineralization rates by a novel <sup>15</sup>N tracing model

SOIL ◽  
2016 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Louise C. Andresen ◽  
Anna-Karin Björsne ◽  
Samuel Bodé ◽  
Leif Klemedtsson ◽  
Pascal Boeckx ◽  
...  
Keyword(s):  
Amino Acids ◽  
Terrestrial Ecosystems ◽  
Organic N ◽  
N Availability ◽  
Simultaneous Quantification ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Use Efficiency ◽  
Soil N Cycle ◽  
Rate Limiting

Abstract. The depolymerization of soil organic matter, such as proteins and (oligo-)peptides, into monomers (e.g. amino acids) is currently considered to be the rate-limiting step for nitrogen (N) availability in terrestrial ecosystems. The mineralization of free amino acids (FAAs), liberated by the depolymerization of peptides, is an important fraction of the total mineralization of organic N. Hence, the accurate assessment of peptide depolymerization and FAA mineralization rates is important in order to gain a better process-based understanding of the soil N cycle. In this paper, we present an extended numerical 15N tracing model Ntrace, which incorporates the FAA pool and related N processes in order to provide a more robust and simultaneous quantification of depolymerization and gross mineralization rates of FAAs and soil organic N. We discuss analytical and numerical approaches for two forest soils, suggest improvements of the experimental work for future studies, and conclude that (i) when about half of all depolymerized peptide N is directly mineralized, FAA mineralization can be as important a rate-limiting step for total gross N mineralization as peptide depolymerization rate; (ii) gross FAA mineralization and FAA immobilization rates can be used to develop FAA use efficiency (NUEFAA), which can reveal microbial N or carbon (C) limitation.

scholarly journals Depolymerization and mineralization – investigating N availability by a novel <sup>15</sup>N tracing model

2016 ◽  
Author(s):  
Louise C. Andresen ◽  
Anna-Karin Björsne ◽  
Samuel Bodé ◽  
Leif Klemedtsson ◽  
Pascal Boeckx ◽  
...  
Keyword(s):  
Amino Acids ◽  
N Mineralization ◽  
Soil Disturbance ◽  
Analytical Models ◽  
N Availability ◽  
Total N ◽  
15N Pool Dilution ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Abstract. Depolymerization of soil organic matter such as proteins and peptides into monomers (e.g. amino acids) is currently thought to be the rate limiting step for N availability in terrestrial N cycles. The mineralization of free amino acids (FAA), liberated by depolymerization of peptides, is an important fraction of the total N mineralization. Accurate assessment 10 of peptide depolymerization and FAA mineralization rates is important in order to gain a better understanding of the N cycle dynamics. Due to the short time span, soil disturbance and unnatural high FAA content during the first few hours after the labelling with the traditional 15N pool dilution experiments, analytical models might overestimate peptide depolymerization rate. In this paper, we present an extended numerical 15N tracing model Ntrace which incorporates the FAA pool and related N processes in order to 1) provide a more robust and coherent estimation of production and mineralization rates of FAAs; 2) 15 and 2) suggest an amino acid N use efficiency (NUEFAA) for soil microbes, which is a more realistic estimation of soil microbial NUE compared to the NUE estimated by analytical methods. We compare analytical and numerical approaches for two forest soils; suggest improvements of the experimental work for future studies; and conclude that: i) FAA mineralization might be as equally an important rate limiting step for gross N mineralization as peptide depolymerization rate is, because about half of all depolymerized peptide N is consecutively being mineralized; and that ii) FAA mineralization and FAA 20 immobilization rates should be used for assessing NUEFAA.

scholarly journals A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Enantiomeric Excess ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

AbstractIn this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg−1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

scholarly journals The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1971 ◽  
Vol 122 (3) ◽  
pp. 267-276 ◽  
Author(s):  
D. C. N. Earl ◽  
Susan T. Hindley
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Donor Site ◽  
Peptide Bond ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Donor And Acceptor ◽  
Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

Substrate-assisted mechanism of catalytic hydrolysis of misaminoacylated tRNA required for protein synthesis fidelity

2019 ◽  
Vol 476 (4) ◽  
pp. 719-732 ◽  
Author(s):  
Mykola M. Ilchenko ◽  
Mariia Yu. Rybak ◽  
Alex V. Rayevsky ◽  
Oksana P. Kovalenko ◽  
Igor Ya. Dubey ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Mechanism ◽  
Thermus Thermophilus ◽  
Extensive Study ◽  
Water Molecules ◽  
Amino Acid Residues ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Mechanism Of Hydrolysis ◽  
Rate Limiting

Abstract d-aminoacyl-tRNA-deacylase (DTD) prevents the incorporation of d-amino acids into proteins during translation by hydrolyzing the ester bond between mistakenly attached amino acids and tRNAs. Despite extensive study of this proofreading enzyme, the precise catalytic mechanism remains unknown. Here, a combination of biochemical and computational investigations has enabled the discovery of a new substrate-assisted mechanism of d-Tyr-tRNATyr hydrolysis by Thermus thermophilus DTD. Several functional elements of the substrate, misacylated tRNA, participate in the catalysis. During the hydrolytic reaction, the 2′-OH group of the А76 residue of d-Tyr-tRNATyr forms a hydrogen bond with a carbonyl group of the tyrosine residue, stabilizing the transition-state intermediate. Two water molecules participate in this reaction, attacking and assisting ones, resulting in a significant decrease in the activation energy of the rate-limiting step. The amino group of the d-Tyr aminoacyl moiety is unprotonated and serves as a general base, abstracting the proton from the assisting water molecule and forming a more nucleophilic ester-attacking species. Quantum chemical methodology was used to investigate the mechanism of hydrolysis. The DFT-calculated deacylation reaction is in full agreement with the experimental data. The Gibbs activation energies for the first and second steps were 10.52 and 1.05 kcal/mol, respectively, highlighting that the first step of the hydrolysis process is the rate-limiting step. Several amino acid residues of the enzyme participate in the coordination of the substrate and water molecules. Thus, the present work provides new insights into the proofreading details of misacylated tRNAs and can be extended to other systems important for translation fidelity.

scholarly journals A multi-enzyme cascade for efficient production of D-p-hydroxyphenylglycine from L-tyrosine

2021 ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Chemical Synthesis ◽  
Specific Activity ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

Abstract In this study, we designed and in vivo reconstructed a novel four-enzyme cascade pathway for the production of D-HPG, a valuable intermediate used to produce β-lactam antibiotics and for fine-chemical synthesis, from L-tyrosine. In this pathway, we identified catalytic conversion of the substrate 4-hydroxyphenylglyoxylic acid by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg− 1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50 g/L L-tyrosine in 24 h with 92.5% conversion and > 99% ee in a 3-L fermenter, representing the highest reported D-HPG titer to date. This four-enzyme cascade provides a novel and effective enzymatic approach to industrial production of D-HPG from cheap amino acids.

Identifiability study of the proteins degradation model, based on ADM1, using simultaneous batch experiments

2006 ◽  
Vol 54 (4) ◽  
pp. 31-39 ◽  
Author(s):  
X. Flotats ◽  
J. Palatsi ◽  
B.K. Ahring ◽  
I. Angelidaki
Keyword(s):  
Amino Acids ◽  
Anaerobic Degradation ◽  
Volatile Fatty Acids ◽  
Batch Experiments ◽  
Degradation Model ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Hydrolysis Process ◽  
Parameter Values ◽  
Rate Limiting

The objective of the present study is to analyse kinetic and stoichiometric parameter values of gelatine anaerobic degradation at thermophilic range, based on an experiment designed to elucidate if volatile fatty acids (VFA) are inhibitors of the hydrolysis process. Results showed that VFA are not inhibiting the hydrolysis process. The ADM1 model adequately expressed the consecutive steps of hydrolysis and acidogenesis, with estimated kinetic values corresponding to a fast acidogenesis and slower hydrolysis. The hydrolysis was found to be the rate limiting step of anaerobic degradation. Estimation of yield coefficients based on the relative initial slopes of VFA profiles obtained in a simple batch experiment produced satisfactory results. From the identification study, it was concluded that it is possible to determine univocally the related kinetic parameter values for protein degradation if the evolution of amino acids is measured in simultaneous batch experiments, with different initial protein and amino acids concentrations.

scholarly journals Biochemical characterisation of a collagenase from Bacillus cereus strain Q1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Isabel J. Hoppe ◽  
Hans Brandstetter ◽  
Esther Schönauer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Triple Helix ◽  
Tertiary Structure ◽  
Helicase Activity ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Biochemical Characterisation ◽  
Rate Limiting ◽  
Unique Amino Acid

AbstractCollagen is the most abundant protein in higher animals and as such it is a valuable source of amino acids and carbon for saprophytic bacteria. Due to its unique amino acid composition and triple-helical tertiary structure it can however only be cleaved by specialized proteases like the collagenases secreted by some bacteria. Among the best described bacterial collagenases are ColG and ColH from Clostridium histolyticum. Many Bacillus species contain homologues of clostridial collagenases, which play a role in some infections caused by B. cereus. Detailed biochemical and enzymatic characterizations of bacillial collagenases are however lacking at this time. In an effort to close this gap in knowledge we expressed ColQ1 from B. cereus strain Q1 recombinantly, investigated its metal dependency and performed peptide, gelatin and collagen degradation assays. Our results show that ColQ1 is a true collagenase, cleaving natively folded collagen six times more efficiently than ColG while at the same time being a similarly effective peptidase as ColH. In both ColQ1 and ColG the rate-limiting step in collagenolysis is the unwinding of the triple-helix. The data suggest an orchestrated multi-domain mechanism for efficient helicase activity.

Ornithine Decarboxylase Activity in Cultured Endothelial Cells Stimulated by Serum, Thrombin and Serotonin

1978 ◽  
Vol 39 (02) ◽  
pp. 496-503 ◽  
Author(s):  
P A D’Amore ◽  
H B Hechtman ◽  
D Shepro
Keyword(s):  
Endothelial Cells ◽  
Ornithine Decarboxylase ◽  
Decarboxylase Activity ◽  
Aortic Endothelial Cells ◽  
Ornithine Decarboxylase Activity ◽  
Rate Limiting Step ◽  
Bovine Aortic Endothelial Cells ◽  
Limiting Step ◽  
Rate Limiting ◽  
Serum Serotonin

SummaryOrnithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10-5 M) or Lilly 11 0140 (10-6M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

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