Determination of the recognition sequence of Mycobacterium smegmatis topoisomerase I on mycobacterial genomic sequences

Mycobacterium tuberculosis (Mtb) DNA gyrase ATPase was the target of a tuberculosis drug discovery program. The low specific activity of the Mtb ATPase prompted the use of Mycobacterium smegmatis (Msm) enzyme as a surrogate for lead generation, since it had 20-fold higher activity. Addition of GyrA or DNA did not significantly increase the activity of the Msm GyrB ATPase, and an assay was developed using GyrB alone. Inhibition of the Msm ATPase correlated well with inhibition of Mtb DNA gyrase supercoiling across three chemical scaffolds, justifying its use. As the IC50 of compounds approached the enzyme concentration, surrogate assays were used to estimate potencies (e.g., the shift in thermal melt of Mtb GyrB, which correlated well with IC50s >10 nM). Analysis using the Morrison equation enabled determination of [Formula: see text]s in the sub-nanomolar range. Surface plasmon resonance was used to confirm these IC50s and measure the Kds of binding, but a fragment of Mtb GyrB had to be used. Across three scaffolds, the dissociation half life, t1/2, of the inhibitor-target complex was ≤8 min. This toolkit of assays was developed to track the potency of enzyme inhibition and guide the chemistry for progression of compounds in a lead identification program.

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Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Journal of Molecular Biology ◽

10.1016/j.jmb.2009.08.064 ◽

2009 ◽

Vol 393 (4) ◽

pp. 788-802 ◽

Cited By ~ 22

Author(s):

Anuradha Gopal Bhat ◽

Majety Naga Leelaram ◽

Shivanand Manjunath Hegde ◽

Valakunja Nagaraja

Keyword(s):

Catalytic Activity ◽

Mycobacterium Smegmatis ◽

Topoisomerase I ◽

Metal Coordination ◽

Distinct Role

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Determination of Basic Probability Assignment Based on Recognition Sequence

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.1049-1050.1171 ◽

2014 ◽

Vol 1049-1050 ◽

pp. 1171-1175

Author(s):

Yan Fei Chen ◽

Xue Zhi Xia ◽

Kui Tu

Keyword(s):

Target Recognition ◽

Evidence Theory ◽

Practical Application ◽

Recognition Sequence ◽

Probability Assignment ◽

Basic Probability Assignment ◽

Recognition Result ◽

Basic Probability ◽

Simulation Results

In some practical application of target recognition with sensors, the sensors will give the recognition sequence of targets, which is more detailed than the single recognition result. How to properly construct the basic probability assignment by the recognition sequence becomes the key to successful application of evidence theory. For the recognition sequence of the target recognition results of general sensor is incomplete, and the importance of the types in the recognition sequence is in descending order, this paper proposes a method to construct weights of recognition sequence, the basic probability assignments constructed by the weights are closer to the real recognition results. Simulation results show that this method is more reasonable and effective than the method of contrast.

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Determination of the Length of Target Recognition Sequence in sgRNA Required for CRISPR Interference

Microbiology and Biotechnology Letters ◽

10.48022/mbl.2111.11003 ◽

2021 ◽

Author(s):

Bumjoon Kim ◽

Byeong Chan Kim ◽

Ho Joung Lee ◽

Sang Jun Lee

Keyword(s):

Target Recognition ◽

Recognition Sequence ◽

Crispr Interference

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Mechanistic insights from structure of Mycobacterium smegmatis topoisomerase I with ssDNA bound to both N- and C-terminal domains

Nucleic Acids Research ◽

10.1093/nar/gkaa201 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4448-4462 ◽

Cited By ~ 2

Author(s):

Nan Cao ◽

Kemin Tan ◽

Xiaobing Zuo ◽

Thirunavukkarasu Annamalai ◽

Yuk-Ching Tse-Dinh

Keyword(s):

Mycobacterium Smegmatis ◽

Topoisomerase I ◽

Physiological Role ◽

Opposite Polarity ◽

Ssdna Binding ◽

Binding Domains ◽

Type Ia ◽

Opposite Strand ◽

Negative Supercoiling ◽

Terminal Domains

Abstract Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.

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Determination of the structure of two novel echistatin variants and comparison of the ability of echistatin variants to inhibit aggregation of platelets from different species

Biochemical Journal ◽

10.1042/bj3050513 ◽

1995 ◽

Vol 305 (2) ◽

pp. 513-520 ◽

Cited By ~ 14

Author(s):

Y L Chen ◽

T F Huang ◽

S W Chen ◽

I H Tsai

Keyword(s):

Platelet Glycoprotein ◽

Medium Size ◽

Recognition Sequence ◽

High Affinity ◽

Cnbr Cleavage ◽

Echis Carinatus ◽

Fluorescent Spectra ◽

Methionine Residues ◽

Aggregation Of Platelets

Two new variants of short disintegrins were purified from the venom of Echis carinatus leakeyi and named echistatin beta and gamma. These proteins were found to be about 85% similar in amino acid sequence to echistatin alpha which has been well studied. The disulphide pattern of echistatin gamma appeared to be identical with that of echistatin alpha. They all contain the adhesive recognition sequence Arg-Gly-Asp (RGD) but inhibit the aggregation of platelets from human and other mammals with different potencies. Echistatin beta and alpha are far more effective on platelets from humans and guinea pigs than those from rabbits and rats whereas echistatin gamma is less discriminating of the platelets of the species tested. This species-dependent platelet sensitivity to echistatin beta and gamma could be attributed to the variations in residues 15, 21, 22 and 27, which are close to or within the RGD loop, rather than to the C-terminal variations after residue 46. Taking advantage of the presence of methionine residues flanking both sides of the ARGDDM motif in echistatin gamma, we deleted this hexapeptide by CNBr cleavage to produce des-(23-28)-echistatin gamma. The modified protein showed c.d. and fluorescent spectra grossly similar to the intact echistatin but its antiplatelet potency decreased more than 200-fold. We thus propose that a favourable conformation of the RGD region is responsible mainly for the high-affinity binding of echistatin to the platelet glycoprotein IIb-IIIa as shown previously for the binding of medium-size disintegrin.

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