Site-specific mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium. Changing arginine 179 to leucine alters the reciprocal transmission of substrate-induced conformational changes between the alpha and beta 2 subunits.

Kawasaki, Haruhiko; Bauerle, Ronald; Zon, Gerald; Ahmed, Salman; Miles, Edith Wilson

doi:10.1016/s0021-9258(18)61017-8

Cited by 120 publications

(95 citation statements)

References 34 publications

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“…E. coli strain CB149 38 harboring the expression vector pEBA10 39 was used to overexpress the α 2 β 2 TRPS complex from Salmonella enterica serovar typhimurium (α 2 β 2 TRPS complex). A scrape from the glycerol stock was used to inoculate 50 ml fresh and sterile Luria Bertani culture medium (LB: 0.5% yeast extract, 1.0% peptone, and 1.0% sodium chloride) supplemented with 50 μg/ml ampicillin and amplified overnight at 37 C and 200rpm 1 .…”

Section: Recombinant Protein Expression and Purificationmentioning

confidence: 99%

Discovery of antimicrobial agent targeting tryptophan synthase

Bosken

Hilario

et al. 2021

Protein Science

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Section: Recombinant Protein Expression and Purificationmentioning

confidence: 99%

Discovery of antimicrobial agent targeting tryptophan synthase

Bosken

Hilario

et al. 2021

Protein Science

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“…Replacing the tryptophan carboxylate with a thiocarboxylate is expected to favor formation of 20 over 5 if the reaction proceeds via Mechanism A because sulfur will stabilize 10 (OH bond is 18 kcal/mol stronger than the SH bond). In contrast, for Mechanism B, this substrate modification will retard the indole carboxylation because of thioformate radical (19) stabilization by the sulfur. This will facilitate the trapping of 14.…”

mentioning

confidence: 94%

Mechanistic Studies on Tryptophan Lyase (NosL): Identification of Cyanide as a Reaction Product

2018

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Tryptophan lyase (NosL) catalyzes the formation of 3-methylindole-2-carboxylic acid and 3-methylindole from l-tryptophan. In this paper, we provide evidence supporting a formate radical intermediate and demonstrate that cyanide is a byproduct of the NosL-catalyzed reaction with l-tryptophan. These experiments require a major revision of the NosL mechanism and uncover an unanticipated connection between NosL and HydG, the radical SAM enzyme that forms cyanide and carbon monoxide from tyrosine during the biosynthesis of the metallo-cluster of the [Fe-Fe] hydrogenase.

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“…2.1.12. Use 200 ng of each plasmid construct (pEBA-10-βQ114A, pEBA-10-βK167T, and pEBA-10-βS377A) to transform competent cells of E. coli expression strain CB149, lacking the trp operon 26 . After successful colony formation, pick one single colony and grow the cells in 5 mL of LB medium containing 50 μg/mL ampicillin.…”

Section: Purification Of the Wild Type Or Mutant Form Of The α 2 β 2 Stts Complexmentioning

confidence: 99%

“…The coding regions of mutant α 2 β 2 StTS complex in pEBA10 were confirmed by DNA sequencing and used to transform E. coli strain CB149 cells 26 . The wild type and mutant form of the α 2 β 2 StTS complex were overexpressed and the recombinant proteins were purified successfully within 1-2 days.…”

Section: Purification Of the Wild Type And Mutant α 2 β 2 Stts Complexmentioning

confidence: 99%

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Hilario

Фан

Mueller

et al. 2020

JoVE

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Structural studies with tryptophan synthase (TS) bienzyme complex (α 2 β 2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated αand isolated β-subunit allowed the purification of high amounts of pure subunits and α 2 β 2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme β-site, sitedirect mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant α 2 β 2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of α 2 β 2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the α 2 β 2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant α 2 β 2 StTS complexes.

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Site-specific mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium. Changing arginine 179 to leucine alters the reciprocal transmission of substrate-induced conformational changes between the alpha and beta 2 subunits.

Cited by 120 publications

References 34 publications

Discovery of antimicrobial agent targeting tryptophan synthase

Discovery of antimicrobial agent targeting tryptophan synthase

Mechanistic Studies on Tryptophan Lyase (NosL): Identification of Cyanide as a Reaction Product

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

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