Characterization of Alanine Dehydrogenase and Its Effect on Streptomyces coelicolorA3(2) Development in Liquid Culture

2019 ◽  
Vol 29 (1-6) ◽  
pp. 57-65
Author(s):  
Arie Van Wieren ◽  
Ryan Cook ◽  
Sudipta Majumdar
Keyword(s):  
Enzyme Activity ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Lag Phase ◽  
Phase Growth ◽  
Carbon And Nitrogen ◽  
Alanine Dehydrogenase ◽  
Essential Enzyme ◽  
Deamination Reaction

<i>Streptomyces</i>, the most important group of industrial microorganisms, is harvested in liquid cultures for the production of two-thirds of all clinically relevant secondary metabolites. It is demonstrated here that the growth of <i>Streptomyces coelicolor</i> A3(2) is impacted by the deletion of the alanine dehydrogenase (ALD), an essential enzyme that plays a central role in the carbon and nitrogen metabolism. A long lag-phase growth followed by a slow exponential growth of <i>S. coelicolor</i> due to ALD gene deletion was observed in liquid yeast extract mineral salt culture. The slow lag-phase growth was replaced by the normal wild-type like growth by ALD complementation engineering. The ALD enzyme from <i>S. coelicolor</i> was also heterologously cloned and expressed in <i>Escherichia coli</i> for characterization. The optimum enzyme activity for the oxidative deamination reaction was found at 30°C, pH 9.5 with a catalytic efficiency, k<sub>cat</sub>/K<sub>M</sub>, of 2.0 ± 0.1 mM<sup>–1</sup> s<sup>–1</sup>. The optimum enzyme activity for the reductive amination reaction was found at 30°C, pH 9.0 with a catalytic efficiency, k<sub>cat</sub>/K<sub>M</sub>, of 1.9 ± 0.1 mM<sup>–1</sup> s<sup>–1</sup>.

Erythrocyte Adenylate Kinase Deficiency: First In-Depth Biochemical Characterization of the Y164C Mutant Enzyme Causing Hemolytic Anemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3602-3602
Author(s):  
Patrizia Abrusci ◽  
Laurent R. Chiarelli ◽  
Elisa Fermo ◽  
Alessandro Galizzi ◽  
Alberto Zanella ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Hemolytic Anemia ◽  
Adenylate Kinase ◽  
Genetic Disorder ◽  
Catalytic Efficiency ◽  
Mutant Enzyme ◽  
Heat Inactivation ◽  
Mutant Form ◽  
Recessive Trait

Abstract Adenylate kinases (AK, EC 2.7.4.3) are monomeric enzymes that catalyze the reversible transfer of the γ-phosphate group from a phosphate donor (normally ATP) to a phosphate acceptor (normally AMP) releasing two molecules of nucleoside diphosphates (ADP). Therefore, the AK enzymes play a crucial role in the synthesis of nucleotides that are required for a variety of cellular metabolic processes. At present, six isoforms of AK with different tissue localization and substrate specificity have been characterized in man. AK1 is a cytosolic enzyme, mainly expressed in skeletal muscle, brain and erythrocytes. Adenylate kinase deficiency is a rare red blood cell enzymopathy causing hemolysis; the genetic disorder is transmitted as an autosomal recessive trait. To date 7 different mutations have been reported. To analyze the mutations at the protein level, we have undertaken the expression, purification and enzymological characterization of the human AK1 in the wild-type and Y164C mutant form. The cDNA encoding the AK1 was obtained from a blood sample of a healthy donor, with normal AK1 activity. Maximal expression (about 100–120 mg of AK1 per liter of E.coli culture) was obtained after 6 hours of induction with 0.5 mM IPTG at 37°C. The recombinant enzyme was purified to homogeneity following a simple procedure. The specific activity was greater than 2000 U/mg. AK1 exhibited a relatively high stability (T50 after 10 min of incubation, 53°C). Moreover, Mg-ATP protected enzyme activity from heat inactivation (T50 about 20 degrees higher). The catalytic efficiency values towards ATP and AMP were essentially identical and similar to that of the authentic enzyme. The Y164C mutation has been found in an Italian patient with congenital hemolytic anemia and undetectable erythrocyte AK1 activity. By site directed-mutagenesis we have produced the Y164C mutant protein and we have performed the first in-depth functional analysis of AK1 causing hemolytic anemia. The mutant enzyme essentially exhibited Km values unchanged, but drastic reductions of the catalytic efficiency towards both substrates (2 orders of magnitude). The Y164C mutant displayed little alterations in the thermostability properties. However, Mg-ATP failed to protect the enzyme activity from heat inactivation. Our data strongly suggest that the absence of AK1 activity observed in the patient homozygous for the Y164C mutation is due to inability of the enzyme to undergo conformational changes essential for the catalytic cycle.

scholarly journals Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Min Li ◽  
Zhi-Jun Zhang ◽  
Xu-Dong Kong ◽  
Hui-Lei Yu ◽  
Jiahai Zhou ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Engineering ◽  
Directed Evolution ◽  
Specific Activity ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Carbonyl Reductase ◽  
Triple Mutant ◽  
Engineering Strategy ◽  
Substrate Tolerance

ABSTRACT Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/I158V/P168S, whose thermostability (T 50 15, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C 50 15, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter−1 day−1. These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor. IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.

Heparin and Low Molecular Weight Heparins Inhibit Prothrombinase Formation but not its Activity in Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 862-868 ◽  
Author(s):  
Frederick A Ofosu ◽  
J C Lormeau ◽  
Sharon Craven ◽  
Lori Dewar ◽  
Noorildan Anvari
Keyword(s):  
Factor Xa ◽  
Catalytic Efficiency ◽  
Thrombin Inhibitor ◽  
Lag Phase ◽  
Factor V ◽  
Factor X ◽  
Normal Plasma ◽  
Plasma Factor ◽  
Prothrombin Activation ◽  
Plasma Heparin

SummaryFactor V activation is a critical step preceding prothrombinase formation. This study determined the contributions of factor Xa and thrombin, which activate purified factor V with similar catalytic efficiency, to plasma factor V activation during coagulation. Prothrombin activation began without a lag phase after a suspension of coagulant phospholipids, CaCl2, and factor Xa was added to factor X-depleted plasma. Hirudin, a potent thrombin inhibitor, abrogated prothrombin activation initiated with 0.5 and 1.0 nM factor Xa, but not with 5 nM factor Xa. In contrast, hirudin did not abrogate prothrombin activation in plasmas pre-incubated with 0.5,1.0 or 5 nM α-thrombin for 10 s followed by the coagulant suspension containing 0.5 nM factor Xa. Thus, thrombin activates plasma factor V more efficiently than factor Xa. At concentrations which doubled the clotting time of contact-activated normal plasma, heparin and three low Mr heparins also abrogated prothrombin activation initiated with 0.5 nM factor Xa, but not with 5 nM factor Xa. If factor V in the factor X-depleted plasma was activated (by pre-incubation with 10 nM a-thrombin for 60 s) before adding 0.5,1.0, or 5 nM factor Xa, neither hirudin nor the heparins altered the rates of prothrombin activation. Thus, none of the five anticoagulants inactivates prothrombinase. When 5 or 10 pM relipidated r-human tissue factor and CaCl2 were added to normal plasma, heparin and the three low Mr heparins delayed the onset of prothrombin activation until the concentration of factor Xa generated exceeded 1 nM, and they subsequently inhibited prothrombin activation to the same extent. Thus, hirudin, heparin and low Mr heparins suppress prothrombin activation solely by inhibiting prothrombinase formation.

scholarly journals An Electrochemical Approach to Follow and Evaluate the Kinetic Catalysis of Ricin on hsDNA

Life ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 405
Author(s):  
George Oliveira ◽  
José Maurício Schneedorf
Keyword(s):  
Catalytic Efficiency ◽  
Ricin Toxin ◽  
Wave Voltammetry ◽  
Progress Curve ◽  
Electrochemical Approach ◽  
Electro Oxidation ◽  
Castor Seeds ◽  
Identification And Characterization ◽  
Medical Countermeasures

International authorities classify the ricin toxin, present in castor seeds, as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin are considered the first actions for its risk assessment during a suspected exposure, parallel to the development of therapeutic and medical countermeasures. In this study, we report the kinetic analysis of electro-oxidation of adenine released from hsDNA by the catalytic action of ricin by square wave voltammetry. The results suggest that ricin-mediated adenine release exhibited an unusual kinetic profile, with a progress curve controlled by the accumulation of the product and the values of the kinetic constants of 46.6 µM for Km and 2000 min−1 for kcat, leading to a catalytic efficiency of 7.1 × 105 s−1 M−1.

scholarly journals In vitro conversion of proapoprotein A-I to apoprotein A-I. Partial characterization of an extracellular enzyme activity.

1983 ◽  
Vol 258 (19) ◽  
pp. 11430-11433 ◽  
Author(s):  
C Edelstein ◽  
J I Gordon ◽  
K Toscas ◽  
H F Sims ◽  
A W Strauss ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Extracellular Enzyme ◽  
Extracellular Enzyme Activity ◽  
Partial Characterization
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