scholarly journals How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

2014 ◽  
Vol 458 (3) ◽  
pp. 449-458 ◽  
Author(s):  
Lisa Bowman ◽  
Lindsey Flanagan ◽  
Paul K. Fyfe ◽  
Alison Parkin ◽  
William N. Hunter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Large Subunit ◽  
Small Subunit ◽  
Oxygen Tolerance

A hydrogenase consists of two subunits: a large and a small subunit. In the present study, amino acids from the large subunit were found to influence a cofactor in the small subunit, such that they help to confer oxygen-tolerance to the enzyme.

The study of plant phylogeny using amino acid sequences of Ribulose-1,5-Bisphosphate carboxylase. I. Patterns of variability

1983 ◽  
Vol 31 (4) ◽  
pp. 395 ◽  
Author(s):  
PG Martin ◽  
AC Jennings
Keyword(s):  
Amino Acids ◽  
Large Subunit ◽  
Small Subunit ◽  
Amino Acid Sequences ◽  
Protein Sequencing ◽  
Ribulose Bisphosphate Carboxylase ◽  
Bisphosphate Carboxylase ◽  
Plant Phylogeny ◽  
Variable Site ◽  
N Terminus

Ribulose bisphosphate carboxylase has been prepared from 50 species of angiosperms from 16 diverse families. In 35 preparations, well known 'bland leaf' methods were used but 15 species had 'pungent leaves' and for these a new preparative method is described. Automatic methods have been used to obtain N-terminal sequences (40 amino acids) of the small subunit (SSU) from all 50 species and the pattern of variability is discussed: 26 of 40 positions are variable to a degree similar to that found in plastocyanin and plant cytochrome c, i.e, an average of 3.7 different amino acids per variable site. These results, and the fact that sufficient protein can be obtained from 100 g of leaves, make a widespread phylogenetic survey of angiosperm SSU feasible and it is claimed that the method is at least as practicable as nucleic acid sequencing. A limited amount of sequencing has been carried out on the large subunit (LSU) but its low variability discourages a protein sequencing survey. Implications for gene structure and function are discussed and evidence is given that active LSU is derived from a precursor with 14 additional amino acids at the N-terminus. In SSU, variability of the two N- terminal amino acids suggests that they are not involved in the signals for removal of either the transit peptide or, in the RNA, of the intron, excision of one end of which depends on the codons for the invariable amino acids at positions 3 and 4. Evidence is also given that if the N-terminus of SSU is methionine, as is common, then it is modified and associated with a 'frayed' N-terminus.

scholarly journals The Epstein-Barr Virus (EBV) Deubiquitinating Enzyme BPLF1 Reduces EBV Ribonucleotide Reductase Activity

2009 ◽  
Vol 83 (9) ◽  
pp. 4345-4353 ◽  
Author(s):  
Christopher B. Whitehurst ◽  
Shunbin Ning ◽  
Gretchen L. Bentz ◽  
Florent Dufour ◽  
Edward Gershburg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ribonucleotide Reductase ◽  
Active Site ◽  
Epstein Barr Virus ◽  
Reductase Activity ◽  
Large Subunit ◽  
Small Subunit ◽  
Deubiquitinating Enzyme ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT A newly discovered virally encoded deubiquitinating enzyme (DUB) is strictly conserved across the Herpesviridae. Epstein-Barr virus (EBV) BPLF1 encodes a tegument protein (3,149 amino acids) that exhibits deubiquitinating (DUB) activity that is lost upon mutation of the active-site cysteine. However, targets for the herpesviral DUBs have remained elusive. To investigate a predicted interaction between EBV BPLF1 and EBV ribonucleotide reductase (RR), a functional clone of the first 246 N-terminal amino acids of BPLF1 (BPLF1 1-246) was constructed. Immunoprecipitation verified an interaction between the small subunit of the viral RR2 and BPLF1 proteins. In addition, the large subunit (RR1) of the RR appeared to be ubiquitinated both in vivo and in vitro; however, ubiquitinated forms of the small subunit, RR2, were not detected. Ubiquitination of RR1 requires the expression of both subunits of the RR complex. Furthermore, coexpression of RR1 and RR2 with BPLF1 1-246 abolishes ubiquitination of RR1. EBV RR1, RR2, and BPLF1 1-246 colocalized to the cytoplasm in HEK 293T cells. Finally, expression of enzymatically active BPLF1 1-246 decreased RR activity, whereas a nonfunctional active-site mutant (BPLF1 C61S) had no effect. These results indicate that the EBV deubiquitinating enzyme interacts with, deubiquitinates, and influences the activity of the EBV RR. This is the first verified protein target of the EBV deubiquitinating enzyme.

Microbial hydrogen splitting in the presence of oxygen

2013 ◽  
Vol 41 (5) ◽  
pp. 1317-1324 ◽  
Author(s):  
Matthias Stein ◽  
Sandeep Kaur-Ghumaan
Keyword(s):  
Active Site ◽  
Large Subunit ◽  
Spectroscopic Studies ◽  
Small Subunit ◽  
Reduced Form ◽  
Cysteine Residues ◽  
Binding Motif ◽  
Oxygen Tolerance ◽  
Symmetry Approach ◽  
Long Time

The origin of the tolerance of a subclass of [NiFe]-hydrogenases to the presence of oxygen was unclear for a long time. Recent spectroscopic studies showed a conserved active site between oxygen-sensitive and oxygen-tolerant hydrogenases, and modifications in the vicinity of the active site in the large subunit could be excluded as the origin of catalytic activity even in the presence of molecular oxygen. A combination of bioinformatics and protein structural modelling revealed an unusual co-ordination motif in the vicinity of the proximal Fe–S cluster in the small subunit. Mutational experiments confirmed the relevance of two additional cysteine residues for the oxygen-tolerance. This new binding motif can be used to classify sequences from [NiFe]-hydrogenases according to their potential oxygen-tolerance. The X-ray structural analysis of the reduced form of the enzyme displayed a new type of [4Fe–3S] cluster co-ordinated by six surrounding cysteine residues in a distorted cubanoid geometry. The unusual electronic structure of the proximal Fe–S cluster can be analysed using the broken-symmetry approach and gave results in agreement with experimental Mößbauer studies. An electronic effect of the proximal Fe–S cluster on the remote active site can be detected and quantified. In the oxygen-tolerant hydrogenases, the hydride occupies an asymmetric binding position in the Ni-C state. This may rationalize the more facile activation and catalytic turnover in this subclass of enzymes.

Amino acids 39–456 of the large subunit and 210–262 of the small subunit constitute the minimal functionally interacting fragments of the unusual heterodimeric topoisomerase IB of Leishmania

2007 ◽  
Vol 409 (2) ◽  
pp. 481-489 ◽  
Author(s):  
Somdeb Bosedasgupta ◽  
Benu Brata Das ◽  
Souvik Sengupta ◽  
Agneyo Ganguly ◽  
Amit Roy ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Large Subunit ◽  
Small Subunit ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Subunit Interaction ◽  
Unusual Structure ◽  
Topoisomerase Ib ◽  
Antisense Constructs

The unusual, heterodimeric topoisomerase IB of Leishmania shows functional activity upon reconstitution of the DNA-binding large subunit (LdTOPIL; or L) and the catalytic small subunit (LdTOPIS; or S). In the present study, we generated N- and C-terminal-truncated deletion constructs of either subunit and identified proteins LdTOPIL39–456 (lacking amino acids 1–39 and 457–635) and LdTOPIS210–262 (lacking amino acids 1–210) as the minimal interacting fragments. The interacting region of LdTOPIL lies between residues 40–99 and 435–456, while for LdTOPIS it lies between residues 210–215 and 245–262. The heterodimerization between the two fragments is weak and therefore co-purified fragments showed reduced DNA binding, cleavage and relaxation properties compared with the wild-type enzyme. The minimal fragments could complement their respective wild-type subunits inside parasites when the respective subunits were down-regulated by transfection with conditional antisense constructs. Site-directed mutagenesis studies identify Lys455 of LdTOPIL and Asp261 of LdTOPIS as two residues involved in subunit interaction. Taken together, the present study provides crucial insights into the mechanistic details for understanding the unusual structure and inter-subunit co-operativity of this heterodimeric enzyme.

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.

Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

Theileria parva ribosomal internal transcribed spacer sequences exhibit extensive polymorphism and mosaic evolution: application to the characterization of parasites from cattle and buffalo

Parasitology ◽  
1999 ◽  
Vol 118 (6) ◽  
pp. 541-551 ◽  
Author(s):  
N. E. COLLINS ◽  
B. A. ALLSOPP
Keyword(s):  
Genetic Recombination ◽  
Large Subunit ◽  
Small Subunit ◽  
Internal Transcribed Spacers ◽  
Rrna Genes ◽  
Gene Pools ◽  
Theileria Parva ◽  
Causative Agents ◽  
Internal Transcribed Spacer Sequences

We sequenced the rRNA genes and internal transcribed spacers (ITS) of several Theileria parva isolates in an attempt to distinguish between the causative agents of East coast fever and Corridor disease. The small subunit (SSU) and large subunit (LSU) rRNA genes from a cloned T. p. lawrencei parasite were sequenced; the former was identical to that of T. p. parva Muguga, and there were minor heterogeneities in the latter. The 5·8S gene sequences of 11 T. parva isolates were identical, but major differences were found in the ITS. Six characterization oligonucleotides were designed to hybridize within the variable ITS1 region; 93·5% of T. p. parva isolates examined were detected by probe TPP1 and 81·8% of T. p. lawrencei isolates were detected by TPL2 and/or TPL3a. There was no absolute distinction between T. p. parva and T. p. lawrencei and the former hybridized with fewer of the probes than did the latter. It therefore seems that a relatively homogenous subpopulation of T. parva has been selected in cattle from a more diverse gene pool in buffalo. The ITSs of both T. p. parva and T. p. lawrencei contained different combinations of identifiable sequence segments, resulting in a mosaic of segments in any one isolate, suggesting that the two populations undergo genetic recombination and that their gene pools are not completely separate.

scholarly journals Domains for protein-protein interactions at the N and C termini of the large subunit of bacteriophage lambda terminase.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 477-484
Author(s):  
W F Wu ◽  
S Christiansen ◽  
M Feiss
Keyword(s):  
Protein Interactions ◽  
Ternary Complex ◽  
Large Subunit ◽  
Small Subunit ◽  
Functional Domain ◽  
Binary Complex ◽  
Protein Protein Interactions ◽  
Related Phage ◽  
Carboxy Terminal ◽  
Lambda Dna

Abstract The large subunit of phage lambda terminase, gpA, the gene product of the phage A gene, interacts with the small subunit, gpNul, to form functional terminase. Terminase binds to lambda DNA at cosB to form a binary complex. The terminase:DNA complex binds a prohead to form a ternary complex. Ternary complex formation involves an interaction of the prohead with gpA. The amino terminus of gpA contains a functional domain for interaction with gpNul, and the carboxy-terminal 38 amino acids of gpA contain a functional domain for prohead binding. This information about the structure of gpA was obtained through the use of hybrid phages resulting from recombination between lambda and the related phage 21. lambda and 21 encode terminases that are analogous in structural organization and have ca. 60% sequence identity. In spite of these similarities, lambda and 21 terminases differ in specificity for DNA binding, subunit assembly, and prohead binding. A lambda-21 hybrid phage produces a terminase in which one of the subunits is chimeric and had recombinant specificities. In the work reported here; a new hybrid, lambda-21 hybrid 67, is characterized. lambda-21 hybrid 67 is the result of a crossover between lambda and 21 in the large subunit genes, such that the DNA from the left chromosome end is from 21, including cosB phi 21, the 1 gene, and the first 48 codons for the 2 gene. The rest of the hybrid 67 chromosome is lambda DNA, including 593 codons of the A gene. The chimeric gp2/A of hybrid 67 binds gp1 to form functional terminase.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The Evolution of Life Modes in Stictidaceae, with Three Novel Taxa

2021 ◽  
Vol 7 (2) ◽  
pp. 105
Author(s):  
Vinodhini Thiyagaraja ◽  
Robert Lücking ◽  
Damien Ertz ◽  
Samantha C. Karunarathna ◽  
Dhanushka N. Wanasinghe ◽  
...  
Keyword(s):  
New Species ◽  
Large Scale ◽  
Sequence Data ◽  
Large Subunit ◽  
Monte Carlo Sampling ◽  
Small Subunit ◽  
Sensu Stricto ◽  
Internal Transcribed Spacers ◽  
Character State ◽  
Phenotypic Data

Ostropales sensu lato is a large group comprising both lichenized and non-lichenized fungi, with several lineages expressing optional lichenization where individuals of the same fungal species exhibit either saprotrophic or lichenized lifestyles depending on the substrate (bark or wood). Greatly variable phenotypic characteristics and large-scale phylogenies have led to frequent changes in the taxonomic circumscription of this order. Ostropales sensu lato is currently split into Graphidales, Gyalectales, Odontotrematales, Ostropales sensu stricto, and Thelenellales. Ostropales sensu stricto is now confined to the family Stictidaceae, which includes a large number of species that are poorly known, since they usually have small fruiting bodies that are rarely collected, and thus, their taxonomy remains partly unresolved. Here, we introduce a new genus Ostropomyces to accommodate a novel lineage related to Ostropa, which is composed of two new species, as well as a new species of Sphaeropezia, S. shangrilaensis. Maximum likelihood and Bayesian inference analyses of mitochondrial small subunit spacers (mtSSU), large subunit nuclear rDNA (LSU), and internal transcribed spacers (ITS) sequence data, together with phenotypic data documented by detailed morphological and anatomical analyses, support the taxonomic affinity of the new taxa in Stictidaceae. Ancestral character state analysis did not resolve the ancestral nutritional status of Stictidaceae with confidence using Bayes traits, but a saprotrophic ancestor was indicated as most likely in a Bayesian binary Markov Chain Monte Carlo sampling (MCMC) approach. Frequent switching in nutritional modes between lineages suggests that lifestyle transition played an important role in the evolution of this family.

Morphometrics and molecular analysis ofOzolaimus linstowin. sp. (Oxyuroidea: Pharyngodonidae) from the green lizardIguana iguana

2015 ◽  
Vol 90 (2) ◽  
pp. 186-198 ◽  
Author(s):  
S.V. Malysheva
Keyword(s):  
Body Size ◽  
Large Subunit ◽  
Buccal Capsule ◽  
Small Subunit ◽  
Present Species ◽  
Lsu Rdna ◽  
Adult Males ◽  
Spicule Length ◽  
Males And Females ◽  
Characteristic Features

AbstractOzolaimus linstowin. sp. is described from the large intestine ofIguana iguanaLinnaeus, 1758 from Mexico. The present species can be easily distinguished fromO. megatyphlonandO. cirratusby the presence of a long and slender pharynx not divided into sections, more similar to the remaining two species,O. monhysteraandO. ctenosauri. Ozolaimus linstowin. sp. can be differentiated fromO. monhysteraby the shorter spicule length and smaller body size of both males and females. Males ofO. linstowin. sp. are morphologically close to those ofO. ctenosauri, but females possess a markedly smaller body size and differ in the organization of the oral cuticular armature. Adult males ofO. linstowin. sp. bear some characteristic features of the J3 juvenile morphology in terms of the cuticular organization of the oral and buccal capsule. Phylogenetic analysis ofO.linstowin. sp. using partial small subunit (SSU) and D2–D3 large subunit (LSU) rDNA shows relationships with several Oxyuridae genera.

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