scholarly journals Role of Asp104 in the SHV β-Lactamase

2006 ◽  
Vol 50 (12) ◽  
pp. 4124-4131 ◽  
Author(s):  
Christopher R. Bethel ◽  
Andrea M. Hujer ◽  
Kristine M. Hujer ◽  
Jodi M. Thomson ◽  
Mark W. Ruszczycky ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Efficiency ◽  
Saturation Mutagenesis ◽  
Amino Acid Residues ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Threefold Increase ◽  
Rate Limiting ◽  
Hydrolysis Of

ABSTRACT Among the TEM-type extended-spectrum β-lactamases (ESBLs), an amino acid change at Ambler position 104 (Glu to Lys) results in increased resistance to ceftazidime and cefotaxime when found with other substitutions (e.g., Gly238Ser and Arg164Ser). To examine the role of Asp104 in SHV β-lactamases, site saturation mutagenesis was performed. Our goal was to investigate the properties of amino acid residues at this position that affect resistance to penicillins and oxyimino-cephalosporins. Unexpectedly, 58% of amino acid variants at position 104 in SHV expressed in Escherichia coli DH10B resulted in β-lactamases with lowered resistance to ampicillin. In contrast, increased resistance to cefotaxime was demonstrated only for the Asp104Arg and Asp104Lys β-lactamases. When all 19 substitutions were introduced into the SHV-2 (Gly238Ser) ESBL, the most significant increases in cefotaxime and ceftazidime resistance were noted for both the doubly substituted Asp104Lys Gly238Ser and the doubly substituted Asp104Arg Gly238Ser β-lactamases. Correspondingly, the overall catalytic efficiency (k cat/Km ) of hydrolysis for cefotaxime was increased from 0.60 ± 0.07 μM−1 s−1 (mean ± standard deviation) for Gly238Ser to 1.70 ± 0.01 μM−1 s−1 for the Asp104Lys and Gly238Ser β-lactamase (threefold increase). We also showed that (i) k 3 was the rate-limiting step for the hydrolysis of cefotaxime by Asp104Lys, (ii) the Km for cefotaxime of the doubly substituted Asp104Lys Gly238Ser variant approached that of the Gly238Ser β-lactamase as pH increased, and (iii) Lys at position 104 functions in an energetically additive manner with the Gly238Ser substitution to enhance catalysis of cephalothin. Based on this analysis, we propose that the amino acid at Ambler position 104 in SHV-1 β-lactamase plays a major role in substrate binding and recognition of oxyimino-cephalosporins and influences the interactions of Tyr105 with penicillins.

Role of intestinal basolateral membrane in absorption of nutrients

1992 ◽  
Vol 263 (3) ◽  
pp. R482-R488 ◽  
Author(s):  
C. Cheeseman
Keyword(s):  
Amino Acid ◽  
Basolateral Membrane ◽  
High Capacity ◽  
Allosteric Modulation ◽  
Transporter Protein ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Dibasic Amino Acid ◽  
Rate Limiting

Organic solutes leave the intestinal epithelium and enter the circulation via specific facilitated carriers located in the basolateral membrane. In the case of glucose it is a low-affinity, high-capacity transport system that can adapt to the carbohydrate content of the diet. Chronic diabetes also promotes the exit of glucose, and in both cases the effect results from an increased density of carriers in the basolateral membrane. In contrast, a rapid upregulation of this system that can be induced within 30 min by hyperglycemia does not involve large changes in the amount of transporter protein. Similarly, the absorptive capacity of the small intestine from some amino acids can be influenced by events occurring at the basolateral membrane. In the case of dibasic amino acid absorption, exit from the epithelium is the rate-limiting step. The activity of the basolateral carrier can be increased almost 10-fold within 60 s by the addition of micromolar concentrations of the neutral amino acid leucine to either the lumen or the plasma. This response does not involve the second messenger adenosine 3',5'-cyclic monophosphate and may represent an allosteric modulation of the carrier. These observations are discussed in relation to the role of the basolateral membrane as a locus for controlling intestinal absorption of organic nutrients.

scholarly journals Carbamate C-N Hydrolase Gene ameH Responsible for the Detoxification Step of Methomyl Degradation in Aminobacter aminovorans Strain MDW-2

2020 ◽  
Vol 87 (1) ◽  
Author(s):  
Wankui Jiang ◽  
Chenfei Zhang ◽  
Qinqin Gao ◽  
Mingliang Zhang ◽  
Jiguo Qiu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Degradation Mechanism ◽  
Catalytic Efficiency ◽  
Acid Sites ◽  
Amino Acid Residues ◽  
Content Type ◽  
Key Enzyme ◽  
A Subunit ◽  
Hydrolysis Of

ABSTRACT Methomyl {bis[1-methylthioacetaldehyde-O-(N-methylcarbamoyl)oximino]sulfide} is a highly toxic oxime carbamate insecticide. Several methomyl-degrading microorganisms have been reported so far, but the role of specific enzymes and genes in this process is still unexplored. In this study, a protein annotated as a carbamate C-N hydrolase was identified in the methomyl-degrading strain Aminobacter aminovorans MDW-2, and the encoding gene was termed ameH. A comparative analysis between the mass fingerprints of AmeH and deduced proteins of the strain MDW-2 genome revealed AmeH to be a key enzyme of the detoxification step of methomyl degradation. The results also demonstrated that AmeH was a functional homodimer with a subunit molecular mass of approximately 34 kDa and shared the highest identity (27%) with the putative formamidase from Schizosaccharomyces pombe ATCC 24843. AmeH displayed maximal enzymatic activity at 50°C and pH 8.5. Km and kcat of AmeH for methomyl were 87.5 μM and 345.2 s−1, respectively, and catalytic efficiency (kcat/Km) was 3.9 μM−1 s−1. Phylogenetic analysis revealed AmeH to be a member of the FmdA_AmdA superfamily. Additionally, five key amino acid residues (162, 164, 191, 193, and 207) of AmeH were identified by amino acid variations. IMPORTANCE Based on the structural characteristic, carbamate insecticides can be classified into oxime carbamates (methomyl, aldicarb, oxamyl, etc.) and N-methyl carbamates (carbaryl, carbofuran, isoprocarb, etc.). So far, research on the degradation of carbamate pesticides has mainly focused on the detoxification step and hydrolysis of their carbamate bond. Several genes, such as cehA, mcbA, cahA, and mcd, and their encoding enzymes have also been reported to be involved in the detoxification step. However, none of these enzymes can hydrolyze methomyl. In this study, a carbamate C-N hydrolase gene, ameH, responsible for the detoxification step of methomyl in strain MDW-2 was cloned and the key amino acid sites of AmeH were investigated. These findings provide insight into the microbial degradation mechanism of methomyl.

Biological treatability of a corn wet mill effluent

2002 ◽  
Vol 45 (12) ◽  
pp. 339-346 ◽  
Author(s):  
G. Eremektar ◽  
O. Karahan-Gul ◽  
F. Germirli-Babuna ◽  
S. Ovez ◽  
H. Uner ◽  
...  
Keyword(s):  
Biological Treatment ◽  
Rate Coefficient ◽  
Activated Sludge Process ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Metabolic Products ◽  
High Level ◽  
Biological Treatability ◽  
Rate Limiting ◽  
Hydrolysis Of

Corn wet mill effluents are studied in terms of their characteristics relevant for biological treatment. They have a high COD of mainly soluble and biodegradable nature, with practically no soluble inert components. They generate a relatively high level of soluble residual metabolic products, which affects the choice of the appropriate biological treatment and favors aerobic activated sludge process. Experimental assessment of process kinetics yields typical values. Hydrolysis of the slowly biodegradable COD, the rate limiting step for the utilization of substrate, is characterized by an overall rate coefficient, which is within the range commonly associated for the hydrolysis of starch.

Daily prolactin pulse inhibits the corpus luteum during lactational quiescence in the marsupial, Macropus eugenii

2013 ◽  
Vol 25 (2) ◽  
pp. 456 ◽  
Author(s):  
L. A. Hinds ◽  
C. H. Tyndale-Biscoe
Keyword(s):  
Corpus Luteum ◽  
Tammar Wallaby ◽  
Marked Contrast ◽  
Previous Conclusion ◽  
Macropus Eugenii ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Progesterone Synthesis ◽  
Rate Limiting

The corpus luteum (CL) of the tammar wallaby is inhibited by prolactin during lactation and seasonal quiescence. In seasonal quiescence a daily transient pulse of prolactin (PRL) of less than 2 h duration is sufficient to maintain inhibition. We investigated whether the same inhibition applies in lactation and, if so, how. Our results show that inhibition of the CL during lactation is maintained by a transient pulse of prolactin once a day. They also show that the minimum time without a PRL pulse for the CL to escape inhibition is more than 48 h and less than 72 h. Nevertheless, some animals had a longer refractory period than 72 h, which was reflected in a longer interval to the progesterone peak and birth. These results support the previous conclusion that PRL exercises its effect on a rate-limiting step in progesterone synthesis and secretion rate from the CL, which precedes any increase in its mass. Therefore, we conclude that the role of PRL is to act as a luteostatic agent, an effect that is in marked contrast to its luteotrophic effect in many eutherian species, including rodents.

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.

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