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 A Novel Spirooxindole Derivative Inhibits the Growth of Leishmania donovani Parasites bothIn VitroandIn Vivoby Targeting Type IB Topoisomerase

2016 ◽  
Vol 60 (10) ◽  
pp. 6281-6293 ◽  
Author(s):  
Sourav Saha ◽  
Chiranjit Acharya ◽  
Uttam Pal ◽  
Somenath Roy Chowdhury ◽  
Kahini Sarkar ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Nuclear Dna ◽  
Large Subunit ◽  
Peritoneal Macrophages ◽  
Small Subunit ◽  
Drug Resistant ◽  
Wild Type ◽  
Content Type ◽  
Fluorescence Studies ◽  
Topoisomerase Ib

ABSTRACTVisceral leishmaniasis is a fatal parasitic disease, and there is an emergent need for development of effective drugs against this neglected tropical disease. We report here the development of a novel spirooxindole derivative,N-benzyl-2,2′α-3,3′,5′,6′,7′,7α,α′-octahydro-2methoxycarbonyl-spiro[indole-3,3′-pyrrolizidine]-2-one (compound 4c), which inhibitsLeishmania donovanitopoisomerase IB (LdTopIB) and kills the wild type as well as drug-resistant parasite strains. This compound inhibits catalytic activity of LdTopIB in a competitive manner. Unlike camptothecin (CPT), the compound does not stabilize the DNA-topoisomerase IB cleavage complex; rather, it hinders drug-DNA-enzyme covalent complex formation. Fluorescence studies show that the stoichiometry of this compound binding to LdTopIB is 2:1 (mole/mole), with a dissociation constant of 6.65 μM. Molecular docking with LdTopIB using the stereoisomers of compound 4c produced two probable hits for the binding site, one in the small subunit and the other in the hinge region of the large subunit of LdTopIB. This spirooxindole is highly cytotoxic to promastigotes ofL. donovaniand also induces apoptosis-like cell death in the parasite. Treatment with compound 4c causes depolarization of mitochondrial membrane potential, formation of reactive oxygen species inside parasites, and ultimately fragmentation of nuclear DNA. Compound 4c also effectively clears amastigote forms of wild-type and drug-resistant parasites from infected mouse peritoneal macrophages but has less of an effect on host macrophages. Moreover, compound 4c showed strong antileishmanial efficacies in the BALB/c mouse model of leishmaniasis. This compound potentially can be used as a lead for developing excellent antileishmanial agents against emerging drug-resistant strains of the parasite.

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 Directed Evolution of Biphenyl Dioxygenase: Emergence of Enhanced Degradation Capacity for Benzene, Toluene, and Alkylbenzenes

2001 ◽  
Vol 183 (18) ◽  
pp. 5441-5444 ◽  
Author(s):  
Hikaru Suenaga ◽  
Mariko Mitsuoka ◽  
Yuko Ura ◽  
Takahito Watanabe ◽  
Kensuke Furukawa
Keyword(s):  
Amino Acids ◽  
Aromatic Hydrocarbons ◽  
Burkholderia Cepacia ◽  
Large Subunit ◽  
Site Directed Mutagenesis ◽  
Biphenyl Dioxygenase ◽  
Pseudomonas Pseudoalcaligenes ◽  
Benzene Toluene ◽  
Related Compounds ◽  
Enhanced Degradation

ABSTRACT Biphenyl dioxygenase (Bph Dox) catalyzes the initial oxygenation of biphenyl and related compounds. Bph Dox is a multicomponent enzyme in which a large subunit (encoded by the bphA1 gene) is significantly responsible for substrate specificity. By using the process of DNA shuffling of bphA1 of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepaciaLB400, a number of evolved Bph Dox enzymes were created. Among them, anEscherichia coli clone expressing chimeric Bph Dox exhibited extremely enhanced benzene-, toluene-, and alkylbenzene-degrading abilities. In this evolved BphA1, four amino acids (H255Q, V258I, G268A, and F277Y) were changed from the KF707 enzyme to those of the LB400 enzyme. Subsequent site-directed mutagenesis allowed us to determine the amino acids responsible for the degradation of monocyclic aromatic hydrocarbons.

scholarly journals Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif

1993 ◽  
Vol 13 (1) ◽  
pp. 123-132
Author(s):  
A D Sharrocks ◽  
H Gille ◽  
P E Shaw
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Amino Acid Peptide ◽  
Serum Response

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

scholarly journals Structural identification of the myo-inositol 1,4,5-trisphosphate-binding domain in rat brain inositol 1,4,5-trisphosphate 3-kinase

1997 ◽  
Vol 326 (1) ◽  
pp. 221-225 ◽  
Author(s):  
Shinji TOGASHI ◽  
Kazunaga TAKAZAWA ◽  
Toyoshi ENDO ◽  
Christophe ERNEUX ◽  
Toshimasa ONAYA
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Apparent Homogeneity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Charged Amino Acid Residue

A series of key amino acids involved in Ins(1,4,5)P3 (InsP3) binding and catalytic activity of rat brain InsP3 3-kinase has been identified. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 280, 125–129]. In this study, newly prepared 5′-deletion and site-directed mutants have been compared both for InsP3 binding and InsP3 3-kinase activity. When the protein was expressed from L259 to R459, the activity was lost but InsP3 binding was conserved. Another deletion mutant that had lost only four amino acids after L259 had lost InsP3 binding, and this finding suggests that these residues (i.e. L259DCK262) are involved in InsP3 binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C261 and K262. The two new enzymes were designated M4 (C261S) and M5 (K262A). M4 showed similar Vmax and Km values for InsP3 and ATP to wild-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin–Sepharose. C-terminal deletion mutants that had lost five, seven or nine amino acids showed a large decrease in InsP3 binding and InsP3 3-kinase activity. One mutant that had lost five amino acids (M2) was purified to apparent homogeneity: Km values for both substrates appeared unchanged but Vmax was decreased approx. 40-fold compared with the wild-type enzyme. The results indicate that (1) a positively charged amino acid residue K262 is essential for InsP3 binding and (2) amino acids at the C-terminal end of the protein are necessary to act as a catalyst in the InsP3 3-kinase reaction.

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.

Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker

2017 ◽  
Vol 95 (6) ◽  
pp. 634-643
Author(s):  
Juliano Alves ◽  
Miguel Garay-Malpartida ◽  
João M. Occhiucci ◽  
José E. Belizário
Keyword(s):  
Amino Acid ◽  
Large Subunit ◽  
Small Subunit ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Caspase 7 ◽  
Caspase Family ◽  
The Impact

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD198↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (kcat/KM) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

Site-directed mutagenesis of farnesyl diphosphate synthase; effect of substitution on the three carboxyl-terminal amino acids

1994 ◽  
Vol 72 (1) ◽  
pp. 75-79 ◽  
Author(s):  
Tanetoshi Koyama ◽  
Kazuhiro Saito ◽  
Kyozo Ogura ◽  
Shusei Obata ◽  
Ayumi Takeshita
Keyword(s):  
Amino Acids ◽  
Bacillus Stearothermophilus ◽  
Farnesyl Diphosphate ◽  
Site Directed Mutagenesis ◽  
Farnesyl Diphosphate Synthase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Directed Mutation ◽  
Catalytic Function ◽  
Terminal Amino

Site-directed mutation was introduced into the gene for the farnesyl diphosphate synthase of Bacillus stearothermophilus. To investigate the significance of the three C-terminal amino acids, where arginine is completely conserved throughout the farnesyl diphosphate synthases of prokaryotes and eukaryotes, three kinds of mutant enzymes, R295V, D296G, and H297L, which have replacements of arginine-295 with valine, aspartate-296 with glycine, and histidine-297 with leucine, respectively, were overproduced and purified to homogeneity. All of the three mutant enzymes showed similar catalytic activities to that of the wild-type enzyme, indicating that the basic amino acids including the conserved arginine in the C-terminal region are not essential for catalytic function. They were also similar to the wild-type enzyme with respect to pH optima, thermostability, reaction product, and kinetic parameters for allylic substrates. However, their Km values for isopentenyl diphosphate are approximately twice that of the wild type.

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.

scholarly journals NUCLEAR MUTATION INCREASES STREPTOMYCIN AND SPECTINOMYCIN SENSITIVITY IN CHLAMYDOMONAS

Genetics ◽  
1978 ◽  
Vol 88 (4) ◽  
pp. 643-650
Author(s):  
Robert W Lee ◽  
Jan A Sapp
Keyword(s):  
Large Subunit ◽  
Small Subunit ◽  
Wild Type ◽  
Resistance Mutations ◽  
Erythromycin Resistance ◽  
Spectinomycin Resistance ◽  
Nuclear Mutation ◽  
Resistant Strains ◽  
Nuclear Locus ◽  
Type Strains

ABSTRACT A spontaneously arising nuclear mutation, ss-1, has been identified in Chlamydomonas reinhardtii that decreases both streptomycin and spectinomycin resistance levels about 10-fold after its introduction into all wild-type, streptomycin-resistant and spectinomycin-resistant strains examined. The mutations for resistance map to nuclear and uniparentally inherited (chloroplast) loci. In contrast, no modification of erythromycin resistance was detected after introducing ss-1 into wild-type strains or into strains carrying nuclear or uniparentally inherited erythromycin-resistance mutations. We suggest that ss-1 affects the small subunit of the chloroplast ribosome because others have shown that streptomycin and spectinomycin resistance in C. reinhardtii are associated with this subunit, whereas erythromycin resistance is associated with the large subunit. ss-1 shows no linkage with the nuclear locus for streptomycin resistance.

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