scholarly journals Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

2019 ◽  
Vol 20 (20) ◽  
pp. 5019 ◽  
Author(s):  
Yu Zhou ◽  
Spencer Whitney
Keyword(s):  
Amino Acids ◽  
Directed Evolution ◽  
Rhodobacter Sphaeroides ◽  
Biochemical Analysis ◽  
Large Subunit ◽  
Amino Acid Substitutions ◽  
Rubp Carboxylase ◽  
T7 Promoter ◽  
Accurate Evaluation

Inaccuracies in biochemically characterizing the amount and CO2-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent Escherichia coli (RDE3) screen to successfully select for the first Rhodobacter sphaeroides Rubisco (RsRubisco) mutant with improved CO2-fixing properties. The RsRubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (kcatC, up 27%), carboxylation efficiency (kcatC/Km for CO2, increased 17%), unchanged CO2/O2 specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of RsRubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (L8S8) assembly. An N-terminal Val-11-Ile substitution did not affect RsRubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on RsRubisco production. This study confirms the feasibility of improving Rubisco by directed evolution using an analytical pipeline that can identify false positives and reliably discriminate carboxylation enhancing amino acids changes from those influencing Rubisco biogenesis (solubility).

scholarly journals Phosphorylation of the Termini of Cauliflower mosaic virus Precapsid Protein Is Important for Productive Infection

2007 ◽  
Vol 20 (6) ◽  
pp. 648-658 ◽  
Author(s):  
Julie Champagne ◽  
Marie-Eve Laliberté-Gagné ◽  
Denis Leclerc
Keyword(s):  
Amino Acids ◽  
Mosaic Virus ◽  
Infectious Clone ◽  
Cauliflower Mosaic Virus ◽  
Productive Infection ◽  
Amino Acid Substitutions ◽  
Symptom Development ◽  
Protein Precursor ◽  
C Terminus

Cauliflower mosaic virus (CaMV) coat protein precursor (pre-CP) has 489 amino acids (p57) and is processed by the viral proteinase into three major forms: p44, p39, and p37. The N- and C-terminal extensions of pre-CP are released during maturation by the virus-encoded proteinase. We showed that these extensions are phosphorylated at several sites by host casein kinase II (CKII). We have identified the phosphorylated amino acids using an in vitro phosphorylation assay and tested the effect of mutation of these sites on viral infectivity. Mutation of serines S66, S68, and S72 to alanine in the N-terminal extension abolished phosphorylation of the protein in vitro. Also, mutation of all S and T residues in the C-terminus (450 to 489) made this region insensitive to CKII. Amino acid substitutions also were introduced into a full-length infectious clone of CaMV. Mutated forms of the virus with S66, S68, and S72 substituted with A or D showed a delay in symptom development and affected the infectivity of the virus. However, a mutant with an A substitution of all the S and T residues of the C-terminal extension of CP was not infectious. These results suggest that phosphorylation of the N- and C-termini of CaMV pre-CP plays an important role in the initiation of viral infection.

scholarly journals Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside

2019 ◽  
Vol 20 (24) ◽  
pp. 6224 ◽  
Author(s):  
Bangqiao Yin ◽  
Qinyan Hui ◽  
Muhammad Kashif ◽  
Ran Yu ◽  
Si Chen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Directed Evolution ◽  
Catalytic Efficiency ◽  
Raw Material ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
The Novel ◽  
Wild Type

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived β-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (kcat/Km of 561.72 mM−1 s−1) toward ρ-nitrophenyl-β-d-glucopyranoside (ρNPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 °C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to β-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel β-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides.

scholarly journals N-terminal Region of the Large Subunit ofLeishmania donovaniBisubunit Topoisomerase I Is Involved in DNA Relaxation and Interaction with the Smaller Subunit

2005 ◽  
Vol 280 (16) ◽  
pp. 16335-16344 ◽  
Author(s):  
Benu Brata Das ◽  
Nilkantha Sen ◽  
Somdeb Bose Dasgupta ◽  
Agneyo Ganguly ◽  
Hemanta K. Majumder
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Topoisomerase I ◽  
Leishmania Donovani ◽  
Biochemical Study ◽  
Large Subunit ◽  
Small Subunit ◽  
Bacterial Cells ◽  
Dna Relaxation

Leishmania donovanitopoisomerase I is an unusual bisubunit enzyme. We have demonstrated earlier that the large and small subunit could be reconstitutedin vitroto show topoisomerase I activity. We extend our biochemical study to evaluate the role of the large subunit in topoisomerase activity. The large subunit (LdTOP1L) shows a substantial degree of homology with the core DNA binding domain of the topoisomerase IB family. Two N-terminal truncation constructs, LdTOP1Δ39L (lacking amino acids 1–39) and LdTOP1Δ99L (lacking amino acids 1–99) of the large subunit were generated and mixed with intact small subunit (LdTOP1S). Our observations reveal that residues within amino acids 1–39 of the large subunit have significant roles in modulating topoisomerase I activity (i.e. in vitroDNA relaxation, camptothecin sensitivity, cleavage activity, and DNA binding affinity). Interestingly, the mutant LdTOP1Δ99LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP1Δ99L was unable to pull down glutathioneS-transferase-LdTOP1S in an Ni2+-nitrilotriacetic acid co-immobilization experiment. For further analysis, we co-expressed LdTOP1L and LdTOP1S inEscherichia coliBL21(DE3)pLysS cells. The lysate shows topoisomerase I activity. Immunoprecipitation revealed that LdTOP1L could interact with LdTOP1S, indicating the subunit interaction in bacterial cells, whereas immunoprecipitation of bacterial lysate co-expressing LdTOP1Δ99L and LdTOP1S reveals that LdTOP1Δ99L was significantly deficient at interacting with LdTOP1S to reconstitute topoisomerase I activity. This study demonstrates that heterodimerization between the large and small subunits of the bisubunit enzyme appears to be an absolute requirement for topoisomerase activity. The residue within amino acids 1–39 from the N-terminal end of the large subunit regulates DNA topology during relaxation by controlling noncovalent DNA binding or by coordinating DNA contacts by other parts of the enzyme.

scholarly journals The CaaX Proteases, Afc1p and Rce1p, Have Overlapping but Distinct Substrate Specificities

2000 ◽  
Vol 20 (12) ◽  
pp. 4381-4392 ◽  
Author(s):  
Cynthia Evans Trueblood ◽  
Victor L. Boyartchuk ◽  
Elizabeth A. Picologlou ◽  
David Rozema ◽  
C. Dale Poulter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Single Amino Acid ◽  
In Vitro Assays ◽  
Sequence Motif ◽  
Amino Acid Substitutions ◽  
In The Wild

ABSTRACT Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a1, the a2, or the X position of the a-factor Ca1a2X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a1 position, V, L, I, C, or M at the a2 position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a1 substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

ADAPTIVE CAPACITY OF EINKORN HUSKLESS IN THE SECOND, THIRD AND SEVENTH RUSSIAN FEDERATION REGIONS

1970 ◽  
pp. 34-38
Author(s):  
S. K. Temirbekova ◽  
M. Sh. Begeulov ◽  
Yu. V. Afanaseva ◽  
I. M. Kulikov ◽  
N. E. Ionova
Keyword(s):  
Amino Acids ◽  
Biochemical Analysis ◽  
Chemical Properties ◽  
First Grade ◽  
Essential Amino Acids ◽  
Wheat Grain ◽  
Volume Yield ◽  
Class 1 ◽  
Group B ◽  
Field And Laboratory Conditions

Biochemical, immunological and physico-chemical properties of an ancient wheat grain – hulless spelt cultivar Gremme are investigated. Biochemical analysis of grain revealed a high content of protein, fiber, macro-and microelements, a rich composition of essential amino acids, which is characteristic of ancient wheat species. Evaluated milling and baking properties of spelt flour. Physical and chemical parameters of spelt grain met the requirements for soft wheat grain class 1: the mass fraction of gluten-38.7 %, the nature of the grain-795 g / l, the number of drops-416 C, the total vitreousness-70 %. However, gluten had an increased stickiness, which is obviously due to the increased content of fiber and gliadin fraction. Grinding of spelt grain was carried out on the aggregate mill installation "Miller 100 Lux" to obtain baking flour of various cultivars. The overall yield of flour of the first grinding was 59.7 %. The highest volume yield (359 cm3) and the best organoleptic properties (total baking score – 3.6 points) were observed in a sample of bread baked from spelt flour that meets the requirements for wheat baking flour of the first grade. Studies have confirmed the possibility of using flour produced from the spelt grain of the Gremme variety for the production of bakery products of increased biological, therapeutic and prophylactic, nutritional value and with a high organoleptic rating. Cereals and flour are very rich in trace elements-manganese, selenium, zinc, potassium, iron, phosphorus, vitamins from group B and B, essential amino acids (biochemical analysis was carried out by the Cherkizovo Center). The cultivar is resistant to drought, heat, excessive moisture. Proved immunological properties to several diseases in the field and laboratory conditions-resistant to enzyme-mycotic seed depletion (EMIS), various types of rust, powdery mildew.

Antimyotoxic Activity of Synthetic Peptides Derived from Bothrops atrox Snake Gamma Phospholipase A2 Inhibitor Selected by Virtual Screening

2019 ◽  
Vol 19 (22) ◽  
pp. 1952-1961 ◽  
Author(s):  
J.C. Sobrinho ◽  
A.F. Francisco ◽  
R. Simões-Silva ◽  
A.M. Kayano ◽  
J.J. Alfonso Ruiz Diaz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Docking ◽  
Synthetic Peptides ◽  
Cell Damage ◽  
Neutralization Assay ◽  
Phospholipase Activity ◽  
Phospholipases A2 ◽  
Catalytically Active ◽  
Bothrops Atrox

Background: Several studies have aimed to identify molecules that inhibit the toxic actions of snake venom phospholipases A2 (PLA2s). Studies carried out with PLA2 inhibitors (PLIs) have been shown to be efficient in this assignment. Objective: This work aimed to analyze the interaction of peptides derived from Bothrops atrox PLIγ (atPLIγ) with a PLA2 and to evaluate the ability of these peptides to reduce phospholipase and myotoxic activities. Methods: Peptides were subjected to molecular docking with a homologous Lys49 PLA2 from B. atrox venom modeled by homology. Phospholipase activity neutralization assay was performed with BthTX-II and different ratios of the peptides. A catalytically active and an inactive PLA2 were purified from the B. atrox venom and used together in the in vitro myotoxic activity neutralization experiments with the peptides. Results: The peptides interacted with amino acids near the PLA2 hydrophobic channel and the loop that would be bound to calcium in Asp49 PLA2. They were able to reduce phospholipase activity and peptides DFCHNV and ATHEE reached the highest reduction levels, being these two peptides the best that also interacted in the in silico experiments. The peptides reduced the myotubes cell damage with a highlight for the DFCHNV peptide, which reduced by about 65%. It has been suggested that myotoxic activity reduction is related to the sites occupied in the PLA2 structure, which could corroborate the results observed in molecular docking. Conclusion: This study should contribute to the investigation of the potential of PLIs to inhibit the toxic effects of PLA2s.

The 1- and 2-Naphthylalanine Analogs of Oxytocin and Vasopressin

1995 ◽  
Vol 60 (12) ◽  
pp. 2170-2177 ◽  
Author(s):  
Zdenko Procházka ◽  
Jiřina Slaninová
Keyword(s):  
Amino Acids ◽  
Solid Phase ◽  
Pressor Test

Solid phase technique on p-methylbenzhydrylamine resin was used for the synthesis of four analogs of oxytocin and four analogs of vasopressin with the non-coded amino acids L- or D- and 1- or 2-naphthylalanine and D-homoarginine. [L-1-Nal2]oxytocin, [D-1-Nal2]oxytocin, [L-2-Nal2]oxytocin, [D-2-Nal2]oxytocin, [L-1-Nal2, D-Har8]vasopressin, [D-1-Nal2, D-Har8]vasopressin, [L-2-Nal2, D-Har8]vasopressin and [D-2-Nal2, D-Har8]vasopressin were synthesized. All eight analogs were found to be uterotonic inhibitors in vitro and in vivo. Analogs with 2-naphthylalanine are stronger inhibitors, particularly in the vasopressin series than the analogs with 1-naphthylalanine. Analogs with 1-naphthylalanine have no activity in the pressor test, analogs with 2-naphthylalanine are weak pressor inhibitors.

Sign in / Sign up

Export Citation Format

Share Document