Allosteric regulation of substrate channeling: Salmonella typhimurium tryptophan synthase

Ghosh, Rajib R.; Hilario, E.; Chang, Chia‐en A.; Mueller, Leonard J.; Dunn, Michael F.

doi:10.3389/fmolb.2022.923042

Cited by 6 publications

(2 citation statements)

References 128 publications

(422 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…bridge with D305 upon substate binding. Furthermore, it is part of the larger COMM domain (β102-β189) involved in the allosteric regulation of the catalytic activities of the αand β-subunits (Schneider et al 1998;Ghosh et al 2022). The corresponding side chain signals may be resolved in this less crowded area of the spectrum and assigned by mutation.…”

Section: Tryptophan Synthasementioning

confidence: 99%

Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR – practical aspects

Bell,

Lindemann,

Gerland

et al. 2024

J Biomol NMR

Self Cite

View full text Add to dashboard Cite

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40–140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20–30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

show abstract

Section: Tryptophan Synthasementioning

confidence: 99%

Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR – practical aspects

Bell,

Lindemann,

Gerland

et al. 2024

J Biomol NMR

Self Cite

View full text Add to dashboard Cite

show abstract

“…The enzyme tryptophan synthase (TS) is pyridoxal PLP-dependent and responsible for the final two steps in the biosynthesis of l -Trp . Although TS is commonly present in various organisms including eubacteria, archaebacteria, protista, fungi, and plantae, it is mostly absent in humans and other mammals.…”

Section: Introductionmentioning

confidence: 99%

A Molecular Dynamics Simulation Study of the Effects of βGln114 Mutation on the Dynamic Behavior of the Catalytic Site of the Tryptophan Synthase

Roy,

Karttunen

2024

J. Chem. Inf. Model.

View full text Add to dashboard Cite

L-tryptophan (l-Trp), a vital amino acid for the survival of various organisms, is synthesized by the enzyme tryptophan synthase (TS) in organisms such as eubacteria, archaebacteria, protista, fungi, and plantae. TS, a pyridoxal 5′-phosphate (PLP)-dependent enzyme, comprises α and β subunits that typically form an α2β2 tetramer. The enzyme’s activity is regulated by the conformational switching of its α and β subunits between the open (T state) and closed (R state) conformations. Many microorganisms rely on TS for growth and replication, making the enzyme and the l-Trp biosynthetic pathway potential drug targets. For instance, Mycobacterium tuberculosis, Chlamydiae bacteria, Streptococcus pneumoniae, Francisella tularensis, Salmonella bacteria, and Cryptosporidium parasitic protozoa depend on l-Trp synthesis. Antibiotic-resistant salmonella strains have emerged, underscoring the need for novel drugs targeting the l-Trp biosynthetic pathway, especially for salmonella-related infections. A single amino acid mutation can significantly impact enzyme function, affecting stability, conformational dynamics, and active or allosteric sites. These changes influence interactions, catalytic activity, and protein–ligand/protein–protein interactions. This study focuses on the impact of mutating the βGln114 residue on the catalytic and allosteric sites of TS. Extensive molecular dynamics simulations were conducted on E(PLP), E(AEX1), E(A–A), and E(C3) forms of TS using the WT, βQ114A, and βQ114N versions. The results show that both the βQ114A and βQ114N mutations increase protein backbone root mean square deviation fluctuations, destabilizing all TS forms. Conformational and hydrogen bond analyses suggest the significance of βGln114 drifting away from cofactor/intermediates and forming hydrogen bonds with water molecules necessary for l-Trp biosynthesis. The βQ114A mutation creates a gap between βAla114 and cofactor/intermediates, hindering hydrogen bond formation due to short side chains and disrupting β-sites. Conversely, the βQ114N mutation positions βAsn114 closer to cofactor/intermediates, forming hydrogen bonds with O3 of cofactors/intermediates and nearby water molecules, potentially disrupting the l-Trp biosynthetic mechanism.

show abstract

Molecular mechanics studies of factors affecting overall rate in cascade reactions: Multi‐enzyme colocalization and environment

Kaushik,

Hung,

Chang

2024

Protein Science

View full text Add to dashboard Cite

Millions of years of evolution have optimized many biosynthetic pathways by use of multi‐step catalysis. In addition, multi‐step metabolic pathways are commonly found in and on membrane‐bound organelles in eukaryotic biochemistry. The fundamental mechanisms that facilitate these reaction processes provide strategies to bioengineer metabolic pathways in synthetic chemistry. Using Brownian dynamics simulations, here we modeled intermediate substrate transportation of colocalized yeast–ester biosynthesis enzymes on the membrane. The substrate acetate ion traveled from the pocket of aldehyde dehydrogenase to its target enzyme acetyl‐CoA synthetase, then the substrate acetyl CoA diffused from Acs1 to the active site of the next enzyme, alcohol‐O‐acetyltransferase. Arranging two enzymes with the smallest inter‐enzyme distance of 60 Å had the fastest average substrate association time as compared with anchoring enzymes with larger inter‐enzyme distances. When the off‐target side reactions were turned on, most substrates were lost, which suggests that native localization is necessary for efficient final product synthesis. We also evaluated the effects of intermolecular interactions, local substrate concentrations, and membrane environment to bring mechanistic insights into the colocalization pathways. The computation work demonstrates that creating spatially organized multi‐enzymes on membranes can be an effective strategy to increase final product synthesis in bioengineering systems.

show abstract

Allosteric regulation of substrate channeling: Salmonella typhimurium tryptophan synthase

Cited by 6 publications

References 128 publications

Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR – practical aspects

Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR – practical aspects

A Molecular Dynamics Simulation Study of the Effects of βGln114 Mutation on the Dynamic Behavior of the Catalytic Site of the Tryptophan Synthase

Molecular mechanics studies of factors affecting overall rate in cascade reactions: Multi‐enzyme colocalization and environment

Contact Info

Product

Resources

About