scholarly journals Characterization of a novel mesophilic CTP-dependent riboflavin kinase and rational engineering to create its thermostable homologs

2021 ◽  
Author(s):  
Yashwant Kumar ◽  
Reman Kumar Singh ◽  
Amrita Brajagopal Hazra
Keyword(s):  
Metal Ion ◽  
Sequence Similarity ◽  
Enzyme Engineering ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
Flavin Mononucleotide ◽  
Wide Range ◽  
Flavin Biosynthesis

ABSTRACTFlavins play a central role in cellular metabolism as molecules that catalyze a wide range of oxidation-reduction reactions in living organisms. Several interesting variations in flavin biosynthesis exist among the domains of life, and the analysis of enzymes on this pathway have put forth many unique structural and mechanistic insights till date. The CTP-dependent riboflavin kinase in archaea is one such example - unlike most kinase enzymes that use adenosine triphosphate to conduct phosphorylation reactions, riboflavin kinases from archaea utilizes cytidine triphosphate (CTP) to phosphorylate riboflavin to produce flavin mononucleotide (FMN). In this study, we present the characterization of a new mesophilic archaeal riboflavin kinase homolog from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic homolog from Methanocaldococcus jannaschii (MjRibK). We reconstitute the activity of the CTP-dependent MmpRibK, determine its kinetic parameters, and analyse the molecular factors that contribute to the uncommon properties of this class of enzymes. Specifically, we probe the flexibility of MmpRibK and MjRibK under varying temperatures and the role of a metal ion for substrate binding and catalysis using molecular dynamics simulation and a series of experiments. Furthermore, based on the high degree of sequence similarity between the mesophilic MmpRibK and the thermophilic MjRibK, we use comparative analysis and site-directed mutagenesis to establish a set of the residues that are responsible for the thermostability of the enzyme without any loss in activity or substrate specificity. Our work contributes to the molecular understanding of flavin biosynthesis in archaea through the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to enzymes, with implications in enzyme engineering and biotechnological applications.

scholarly journals CRYOGLOBULIN TEST AND CRYOGLOBULINEMIA HEPATITIS C-VIRUS RELATED

2016 ◽  
Vol 9 (1) ◽  
pp. e2017007 ◽  
Author(s):  
Umberto Basile
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Confounding Factor ◽  
Underlying Disease ◽  
Clinical Syndrome ◽  
Laboratory Techniques ◽  
Preanalytical Phase ◽  
Wide Range

Cryoglobulins are immunoglobulins that precipitate in serum at temperatures below 37°C and resolubilize upon warming. The clinical syndrome of cryoglobulinemia usually includes purpura, weakness, and arthralgia, but the underlying disease may also contribute other symptoms. Blood samples for cryoglobulin are collected, transported, clotted and spun at 37°C, before the precipitate is allowed to form when serum is stored at 4°C in a Wintrobe tube for at least seven days. The most critical and confounding factor affecting the cryoglobulin test is when the preanalytical phase is not fully completed at 37°C. The easiest way to quantify cryoglobulins is the cryocrit estimate. However, this approach has low accuracy and sensitivity. Furthermore, the precipitate should be resolubilized by warming to confirm that it is truly formed of cryoglobulins. The characterization of cryoglobulins requires the precipitate is several times washed, before performing immunofixation, a technique by which cryoglobulins can be classified depending on the characteristics of the detected immunoglobulins. These features imply a pathogenic role of these molecules which are consequently associated with a wide range of symptoms and manifestations. According to the Brouet classification, Cryoglobulins are grouped into three types by the immunochemical properties of immunoglobulins in the cryoprecipitate. The aim of this paper is to review the major aspects of cryoglobulinemia and the laboratory techniques used to detect and characterize cryoglobulins, taking into consideration the presence and consequences of cryoglobulinemia in Hepatitis C Virus (HCV) infection.

scholarly journals A method to construct the dynamic landscape of a bio-membrane with experiment and simulation

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Albert A. Smith ◽  
Alexander Vogel ◽  
Oskar Engberg ◽  
Peter W. Hildebrand ◽  
Daniel Huster
Keyword(s):  
Molecular Dynamics ◽  
Nmr Relaxation ◽  
Dynamics Simulation ◽  
Comprehensive Description ◽  
Correlation Times ◽  
Wide Range ◽  
Dynamic Landscape ◽  
C Nmr ◽  
Analysis Of Motion

AbstractBiomolecular function is based on a complex hierarchy of molecular motions. While biophysical methods can reveal details of specific motions, a concept for the comprehensive description of molecular dynamics over a wide range of correlation times has been unattainable. Here, we report an approach to construct the dynamic landscape of biomolecules, which describes the aggregate influence of multiple motions acting on various timescales and on multiple positions in the molecule. To this end, we use 13C NMR relaxation and molecular dynamics simulation data for the characterization of fully hydrated palmitoyl-oleoyl-phosphatidylcholine bilayers. We combine dynamics detector methodology with a new frame analysis of motion that yields site-specific amplitudes of motion, separated both by type and timescale of motion. In this study, we show that this separation allows the detailed description of the dynamic landscape, which yields vast differences in motional amplitudes and correlation times depending on molecular position.

Characterization of proteins with Siaα2-3/6Gal-linked glycans from bovine milk and role of their glycans against influenza A virus

2018 ◽  
Vol 9 (10) ◽  
pp. 5198-5208 ◽  
Author(s):  
Hanjie Yu ◽  
Yaogang Zhong ◽  
Zhiwei Zhang ◽  
Xiawei Liu ◽  
Kun Zhang ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Bovine Milk ◽  
Milk Proteins ◽  
Biological Functions ◽  
Wide Range ◽  
Range Of Functions ◽  
Bovine Milk Proteins

The bovine milk proteins have a wide range of functions, but the role of the attached glycans in their biological functions has not been fully understood yet.

Protein Fibrillar Nanopolymers: Molecular-Level Insights into Their Structural, Physical and Mechanical Properties

2015 ◽  
Vol 10 (03) ◽  
pp. 135-156 ◽  
Author(s):  
Valeriya M. Trusova
Keyword(s):  
Protein Misfolding ◽  
Amyloid Fibrils ◽  
Molecular Level ◽  
Physical And Mechanical Properties ◽  
Present Contribution ◽  
Wide Range ◽  
Generic Class ◽  
Protein Fibril

Amyloid fibrils represent a generic class of mechanically strong and stable biomaterials with extremely advantageous properties. Although amyloids were initially associated only with severe neurological disorders, the role of these structures nowadays is shifting from health debilitating to highly beneficial both in biomedical and technological aspects. Intensive involvement of fibrillar assemblies into the wide range of pathogenic and functional processes strongly necessitate the molecular level characterization of the structural, physical and elastic features of protein nanofibrils. In the present contribution, we made an attempt to highlight the up-to-date progress in the understanding of amyloid properties from the polymer physics standpoint. The fundamental insights into protein fibril behavior are essential not only for development of therapeutic strategies to combat the protein misfolding disorders but also for rational and precise design of novel biodegradable protein-based nanopolymers.

Characterization of Lobula Giant Neurons Responsive to Visual Stimuli That Elicit Escape Behaviors in the Crab Chasmagnathus

2007 ◽  
Vol 98 (4) ◽  
pp. 2414-2428 ◽  
Author(s):  
Violeta Medan ◽  
Damián Oliva ◽  
Daniel Tomsic
Keyword(s):  
Visual Memory ◽  
Visual Stimuli ◽  
Visually Guided ◽  
Wide Range ◽  
Large Motion ◽  
Escape Behaviors ◽  
Neuronal Type

In the grapsid crab Chasmagnathus, a visual danger stimulus elicits a strong escape response that diminishes rapidly on stimulus repetition. This behavioral modification can persist for several days as a result of the formation of an associative memory. We have previously shown that a generic group of large motion-sensitive neurons from the lobula of the crab respond to visual stimuli and accurately reflect the escape performance. Additional evidence indicates that these neurons play a key role in visual memory and in the decision to initiate an escape. Although early studies recognized that the group of lobula giant (LG) neurons consisted of different classes of motion-sensitive cells, a distinction between these classes has been lacking. Here, we recorded in vivo the responses of individual LG neurons to a wide range of visual stimuli presented in different segments of the animal's visual field. Physiological characterizations were followed by intracellular dye injections, which permitted comparison of the functional and morphological features of each cell. All LG neurons consisted of large tangential arborizations in the lobula with axons projecting toward the midbrain. Functionally, these cells proved to be more sensitive to single objects than to flow field motion. Despite these commonalities, clear differences in morphology and physiology allowed us to identify four distinct classes of LG neurons. These results will permit analysis of the role of each neuronal type for visually guided behaviors and will allow us to address specific questions on the neuronal plasticity of LGs that underlie the well-recognized memory model of the crab.

scholarly journals Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis

2019 ◽  
Vol 20 (4) ◽  
pp. 916 ◽  
Author(s):  
Diana Humer ◽  
Oliver Spadiut
Keyword(s):  
Catalytic Activity ◽  
Horseradish Peroxidase ◽  
Signal Sequence ◽  
Enzyme Engineering ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Promising Alternative ◽  
Wide Range ◽  
Quadruple Mutant ◽  
Commercial Applications

Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in Escherichia coli as a DsbA fusion protein including a Dsb signal sequence for translocation to the periplasm and a His tag for purification. The missing N-glycosylation results in reduced catalytic activity and thermal stability, therefore enzyme engineering was used to improve these characteristics. The amino acids at four N-glycosylation sites, namely N13, N57, N255 and N268, were mutated by site-directed mutagenesis and combined to double, triple and quadruple enzyme variants. Subsequently, the rHRP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) and biochemically characterized. We found that the quadruple mutant rHRP N13D/N57S/N255D/N268D showed 2-fold higher thermostability and 8-fold increased catalytic activity with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as reducing substrate when compared to the non-mutated rHRP benchmark enzyme.

scholarly journals Characterization of the Hepatitis C Virus NS2/3 Processing Reaction by Using a Purified Precursor Protein

2001 ◽  
Vol 75 (20) ◽  
pp. 9939-9946 ◽  
Author(s):  
Michele Pallaoro ◽  
Armin Lahm ◽  
Gabriella Biasiol ◽  
Mirko Brunetti ◽  
Caterina Nardella ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cleavage Site ◽  
Metal Ion ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Sequence Alignments ◽  
Processing Rate

ABSTRACT The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

scholarly journals Characterization of a Novel Bifunctional Dihydropteroate Synthase/Dihydropteroate Reductase Enzyme from Helicobacter pylori

2007 ◽  
Vol 189 (11) ◽  
pp. 4062-4069 ◽  
Author(s):  
Itay Levin ◽  
Moshe Mevarech ◽  
Bruce A. Palfey
Keyword(s):  
Helicobacter Pylori ◽  
Reducing Power ◽  
Site Directed Mutagenesis ◽  
Flavin Mononucleotide ◽  
Amino Acid Residues ◽  
Dihydropteroate Synthase ◽  
Terminal Domain ◽  
Initiation Of Translation ◽  
H Pylori

ABSTRACT Tetrahydrofolate is a ubiquitous C1 carrier in many biosynthetic pathways in bacteria, importantly, in the biosynthesis of formylmethionyl tRNAfMet, which is essential for the initiation of translation. The final step in the biosynthesis of tetrahydrofolate is carried out by the enzyme dihydrofolate reductase (DHFR). A search of the complete genome sequence of Helicobacter pylori failed to reveal any sequence that encodes DHFR. Previous studies demonstrated that the H. pylori dihydropteroate synthase gene folP can complement an Escherichia coli strain in which folA and folM, encoding two distinct DHFRs, are deleted. It was also shown that H. pylori FolP possesses an additional N-terminal domain that binds flavin mononucleotide (FMN). Homologous domains are found in FolP proteins of other microorganisms that do not possess DHFR. In this study, we demonstrated that H. pylori FolP is also a dihydropteroate reductase that derives its reducing power from soluble flavins, reduced FMN and reduced flavin adenine dinucleotide. We also determined the stoichiometry of the enzyme-bound flavin and showed that half of the bound flavin is exchangeable with the soluble flavins. Finally, site-directed mutagenesis of the most conserved amino acid residues in the N-terminal domain indicated the importance of these residues for the activity of the enzyme as a dihydropteroate reductase.

scholarly journals Role of the ssu and seu Genes of Corynebacterium glutamicum ATCC 13032 in Utilization of Sulfonates and Sulfonate Esters as Sulfur Sources

2005 ◽  
Vol 71 (10) ◽  
pp. 6104-6114 ◽  
Author(s):  
D. J. Koch ◽  
C. Rückert ◽  
D. A. Rey ◽  
A. Mix ◽  
A. Pühler ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Sequence Similarity ◽  
Gene Clusters ◽  
Flavin Mononucleotide ◽  
Gene Products ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Mutant Strains ◽  
Similarity Searches

ABSTRACT Corynebacterium glutamicum ATCC 13032 was found to be able to utilize a broad range of sulfonates and sulfonate esters as sulfur sources. The two gene clusters potentially involved in sulfonate utilization, ssuD1CBA and ssuI-seuABC-ssuD2, were identified in the genome of C. glutamicum ATCC 13032 by similarity searches. While the ssu genes encode proteins resembling Ssu proteins from Escherichia coli or Bacillus subtilis, the seu gene products exhibited similarity to the dibenzothiophene-degrading Dsz monooxygenases of Rhodococcus strain IGTS8. Growth tests with the C. glutamicum wild-type and appropriate mutant strains showed that the clustered genes ssuC, ssuB, and ssuA, putatively encoding the components of an ABC-type transporter system, are required for the utilization of aliphatic sulfonates. In C. glutamicum sulfonates are apparently degraded by sulfonatases encoded by ssuD1 and ssuD2. It was also found that the seu genes seuA, seuB, and seuC can effectively replace ssuD1 and ssuD2 for the degradation of sulfonate esters. The utilization of all sulfonates and sulfonate esters tested is dependent on a novel putative reductase encoded by ssuI. Obviously, all monooxygenases encoded by the ssu and seu genes, including SsuD1, SsuD2, SeuA, SeuB, and SeuC, which are reduced flavin mononucleotide dependent according to sequence similarity, have SsuI as an essential component. Using real-time reverse transcription-PCR, the ssu and seu gene cluster was found to be expressed considerably more strongly during growth on sulfonates and sulfonate esters than during growth on sulfate.

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