Identification and Validation of Aspartic Acid Semialdehyde Dehydrogenase as a New Anti-Mycobacterium  Tuberculosis Target

Aspartate-semialdehyde dehydrogenase (ASADH) of DAP/lysine pathway plays a crucial role in sustainable growth and pathogenicity of Mycobacterium tuberculosis (Mtb) via reductive dephosphorylation of the β-aspartyl phosphate (AP). Inhibition of ASADH through different lead molecules has been gaining high impetus due to its indispensable role in the pathogen’s survival. In the present study, we aimed to decipher the novel lead molecule against Mtb. The AP, a substrate of the DAP/lysine pathway, was used as a template to design new lead molecules to advance the understanding of the molecular inhibition mechanism of Mtb-ASADH. Monodentate and bidentate groups at three different substitution sites of AP were considered to generate a virtual library of new molecules using the combinatorial approach of the LeadGrow module of the VLifeMDS package. These substrate analogs were sifted through ADRXWS drug-likeness descriptors of the module above. Multi-scoring docking was achieved using Biopredicta, Molecular Virtual Docker, and AutoDock Tools. The adopted combinatorial approach yielded 6000 new molecules that reduced to 4979 plausible hits after lead-like filtration. The post-analysis of ADMET and molecular docking exhibited two pro-lead molecules, namely AP0600 and AP0639. The study delineates the substantial understanding of the Mtb-ASADH inhibition mechanism that would undoubtedly accelerate the pace of antitubercular design, thereby gaining more in-depth knowledge to eradicate tuberculosis across the globe.

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The biosynthesis of threonine by mammalian cells: expression of a complete bacterial biosynthetic pathway in an animal cell

Biochemical Journal ◽

10.1042/bj3090999 ◽

1995 ◽

Vol 309 (3) ◽

pp. 999-1007 ◽

Cited By ~ 13

Author(s):

W D Rees ◽

S M Hay

Keyword(s):

Aspartic Acid ◽

Mammalian Cells ◽

Animal Cell ◽

Simian Virus 40 ◽

3T3 Cells ◽

Homoserine Dehydrogenase ◽

Cell Cycle Time ◽

Mammalian Expression ◽

Threonine Synthase ◽

Semialdehyde Dehydrogenase

The coding regions for the Escherichia coli gene for aspartokinase I/homoserine dehydrogenase I (thrA) and the Corynebacterium glutamicum gene for aspartic semialdehyde dehydrogenase (asd) have been subcloned into a Simian Virus 40 (SV40)-based mammalian expression vector. Both enzyme activities are expressed in mouse 3T3 cells after transfer of the corresponding chimaeric gene. The kinetic parameters are similar to those of the native bacterial enzymes, and aspartokinase I/homoserine dehydrogenase I retains its allosteric regulation by threonine. An extract of the cells expressing aspartokinase I/homoserine dehydrogenase I, mixed with one from cells expressing aspartic semialdehyde dehydrogenase, produced homoserine when the mixture was incubated with aspartic acid, ATP and NADPH. The thrA and asd expression cassettes were combined into a single plasmid which, when transfected into 3T3 cells, enabled them to produce homoserine from aspartic acid. Homoserine-producing 3T3 cells were transfected with the plasmid pSVthrB/C (homoserine kinase and threonine synthase) and selected for growth on homoserine. Cell lines isolated from these cells expressed the complete bacterial threonine pathway, were independent of threonine for growth and could be maintained in medium which contained no free threonine. The threonine in the proteins of these cells became enriched in 15N when the culture medium contained [15N]aspartic acid. The production of homoserine and the growth of cells was at a maximum when there was more than 2.5 mM aspartate in the medium. Below this concentration the high Km of aspartokinase limited the flux through the pathway. In the presence of additional aspartic acid the new pathway could sustain a cell cycle time close to that of the same cells cultured in threonine-containing medium.

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DESAIN TAQMAN PROBE SECARA IN SILICO SEBAGAI PENDETEKSI MUTASI PADA KODON 516 GEN rpoB Mycobacterium tuberculosis UNTUK METODE REAL-TIME PCR

Metamorfosa Journal of Biological Sciences ◽

10.24843/metamorfosa.2017.v04.i01.p04 ◽

2017 ◽

Vol 4 (1) ◽

pp. 22

Author(s):

Dek Pueteri Dewi Suryani ◽

Putu Sanna Yustiantara ◽

Sagung Chandra Yowani

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Aspartic Acid ◽

Real Time Pcr ◽

In Silico ◽

Rpob Gene ◽

Taqman Probe ◽

Probe Design ◽

Accession Number ◽

Pcr Method

The aim of this research was to in silico design of TaqMan probe. Design of TaqMan probe were conducted using software Clone Manager Suite 6. As a template, the rpoB gene of M. tuberculosis H37Rv (accession number U12205.1) was used. The results of this research were 8 sequences such as, R516MV-2, R516MV-3, R516MV-4, R516MV-5, R516MV-7, R516MV-8, R516MV-11, R516MV-13. These sequences were met the criteria of TaqMan probe, such as length of nucleotide (23-28 nucleotide), Tm value (72ºC), %GC (50-58%), runs and repeats (?4 bases), dimer structure in accordance to the requirements and does not form hairpin structures. In addition, these sequences were met labeling criteria of TaqMan probe which are including the location of G bases and the number of G-C bases in sequences. Therefore, these sequences could be labeled by FAM (reporter) at 5' end and TAMRA (quencher) at 3' end. The conclusion of this research, the sequences were met the criteria of TaqMan probe. Therefore, it could be targeted to detect mutations at codon 516 with a change of aspartic acid into valine (GAC ? GTC) by using real-time PCR method.

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Cloning and characterization of a gene (msdA) encoding methylmalonic acid semialdehyde dehydrogenase from Streptomyces coelicolor.

Journal of Bacteriology ◽

10.1128/jb.178.2.490-495.1996 ◽

1996 ◽

Vol 178 (2) ◽

pp. 490-495 ◽

Cited By ~ 23

Author(s):

Y X Zhang ◽

L Tang ◽

C R Hutchinson

Keyword(s):

Methylmalonic Acid ◽

Streptomyces Coelicolor ◽

Semialdehyde Dehydrogenase ◽

Acid Semialdehyde

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