Chlorophyllase from Arabidopsis thaliana Reveals an Emerging Model for Controlling Chlorophyll Hydrolysis DOI Creative Commons
Madison Knapp, Minshik Jo,

Courtney L. Henthorn

et al.

ACS Bio & Med Chem Au, Journal Year: 2024, Volume and Issue: 4(6), P. 353 - 370

Published: Nov. 20, 2024

Chlorophyll (Chl) is one of Nature's most complex pigments to biosynthesize and derivatize. This pigment vital for survival also paradoxically toxic if overproduced or released from a protective protein scaffold. Therefore, along with the mass production Chl, organisms invest in mechanisms control its degradation recycling. One important enzyme that involved these latter processes chlorophyllase. employed by numerous photosynthetic hydrolyze phytol tail Chl. Although traditionally thought catalyze first step Chl degradation, recent work suggests chlorophyllase instead during times abiotic stress conditions produce reactive oxygen species. However, molecular details regarding how chlorophyllases are regulated function under such remain enigmatic. Here, we investigate Arabidopsis thaliana isoform AtCLH2 using site-directed mutagenesis, spectrometry, dynamic light scattering, size-exclusion multiangle both steady-state kinetic thermal stability measurements. Through experiments, show exists as monomer solution contains two disulfide bonds. bond putatively maps active site, whereas other links N-terminal Cys residues together. These bonds cleaved chemical protein-based reductants, respectively, integral maintaining activity, stability, substrate scope enzyme. residue oxidation an emerging regulatory strategy controlling hydrolysis pigments.

Language: Английский

Altering Active-Site Loop Dynamics Enhances Standalone Activity of the Tryptophan Synthase Alpha Subunit DOI Creative Commons
Cristina Duran, Thomas Kinateder,

Caroline Hiefinger

et al.

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(22), P. 16986 - 16995

Published: Nov. 2, 2024

The α-subunit (TrpA) of the allosterically regulated bifunctional tryptophan synthase αββα enzyme catalyzes retro-aldol cleavage indole-glycerol phosphate (IGP) to d-glyceraldehyde 3-phosphate (G3P) and indole. activity is highly dependent on β-subunit (TrpB), which regulates activates TrpA for enhanced function. This contrasts with homologous BX1 from Zea mays that can catalyze same reaction as without requiring presence any additional binding partner. In this study, we computationally evaluated compared conformational landscapes ZmBX1 ZmTrpA enzymes. Our results indicate standalone requires modulation dynamics two relevant active-site loops, loop 6 2, need be synchronized accessing catalytically activated closed state IGP cleavage, well open states favoring indole/G3P release. Taking inspiration evolutionary blueprint using our developed correlation-based tool shortest path map focused rate-determining transition leading state, designed a variant named ZmTrpASPM4-L6BX1, displays 163-fold improvement in catalytic efficiency IGP. study showcases importance fine-tuning loops altering improving function, especially those cases change rate determining.

Language: Английский

Citations

4
Advances in the Simulations of Enzyme Reactivity in the Dawn of the Artificial Intelligence Age DOI Creative Commons
Katarzyna Świderek, J. Bertrán, Kirill Zinovjev

et al.

Wiley Interdisciplinary Reviews Computational Molecular Science, Journal Year: 2025, Volume and Issue: 15(1)

Published: Jan. 1, 2025

ABSTRACT The study of natural enzyme catalytic processes at a molecular level can provide essential information for rational design new enzymes, to be applied in more efficient and environmentally friendly industrial processes. use computational tools, combined with experimental techniques, is providing outstanding milestones the last decades. However, apart from complexity associated nature these large flexible biomolecular machines, full catalyzed process involves different physical chemical steps. Consequently, point view, deep understanding every single step requires selection proper technique get reliable, robust useful results. In this article, we summarize techniques their process, including conformational diversity, allostery those steps, as well enzymes. Because impact artificial intelligence all aspects science during years, special attention has been methods based on foundations some selected recent applications.

Language: Английский

Citations

0
Shortest Path Map correlation-based tool for capturing functionally relevant allosteric networks and its application in enzyme design DOI Creative Commons

Esther Pruna Cortada,

Sílvia Osuna

Comptes Rendus Chimie, Journal Year: 2025, Volume and Issue: 28(G1), P. 383 - 395

Published: April 8, 2025

The impact of distal mutations on enzyme design is analogous to the allosteric regulation effect observed in effector binding or substrate transport within heterocomplexes. Building this analogy, we employed molecular dynamics simulations estimate conformational landscape enzymes and developed Shortest Path Map (SPM) tool. This tool identifies key conformationally relevant positions that regulate enzymatic function. In review, highlight various applications SPM method its use as a for investigating effects landscape, characterizing regulation, rationally designing enzymes. A brief description provided together with several examples reported over years (from us other labs) which has been successfully applied.

Language: Английский

Citations

0
Uncovering Hydroxynitrile Lyase Variants with Promiscuous Diastereoselective Nitroaldolase Activity toward the Highly Stereocontrolled Synthesis of Anti β-Nitroalcohols DOI

Ayon Chatterjee,

G. Priyanka,

N. Prakash Prabhu

et al.

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(16), P. 12623 - 12634

Published: Aug. 7, 2024

Diastereoselective Henry reaction (DHR) is an effective direct C–C bond ligation transformation that enables the synthesis of β-nitroalcohols having two contiguous chiral centers. Despite significant applications optically active β-nitroalcohol diastereomers as key intermediates for important pharmaceuticals and biologically molecules, biocatalytic asymmetric remains underdeveloped. Here, we show thatArabidopsis thaliana hydroxynitrile lyase (AtHNL) variants with single amino acid substitution, Y14C Y14A, empower promiscuous DHR a broad synthetic scope toward different aldehydes nitroalkane high enantio- diastereoselectivity (up to >99% ee, de, ic, ∼3470 total turnover number) in production diverse anti (1R,2S)-β-nitroalcohols. displayed remarkable >250-fold increase catalytic efficiency wild-type stereoselective nitroethane addition benzaldehyde. A gram-scale biocatalysis was illustrated using readily available benzaldehyde followed by one-step chemical reduction product prepare l-norephedrine, therapeutic, ee de. The origin probed computational studies isotope labeling experiments. This study describes uncovering providing simple, sustainable, economical platform repertoire anti-β-nitroalcohol structural diversity excellent stereocontrol.

Language: Английский

Citations

1
Chlorophyllase from Arabidopsis thaliana Reveals an Emerging Model for Controlling Chlorophyll Hydrolysis DOI Creative Commons
Madison Knapp, Minshik Jo,

Courtney L. Henthorn

et al.

ACS Bio & Med Chem Au, Journal Year: 2024, Volume and Issue: 4(6), P. 353 - 370

Published: Nov. 20, 2024

Chlorophyll (Chl) is one of Nature's most complex pigments to biosynthesize and derivatize. This pigment vital for survival also paradoxically toxic if overproduced or released from a protective protein scaffold. Therefore, along with the mass production Chl, organisms invest in mechanisms control its degradation recycling. One important enzyme that involved these latter processes chlorophyllase. employed by numerous photosynthetic hydrolyze phytol tail Chl. Although traditionally thought catalyze first step Chl degradation, recent work suggests chlorophyllase instead during times abiotic stress conditions produce reactive oxygen species. However, molecular details regarding how chlorophyllases are regulated function under such remain enigmatic. Here, we investigate Arabidopsis thaliana isoform AtCLH2 using site-directed mutagenesis, spectrometry, dynamic light scattering, size-exclusion multiangle both steady-state kinetic thermal stability measurements. Through experiments, show exists as monomer solution contains two disulfide bonds. bond putatively maps active site, whereas other links N-terminal Cys residues together. These bonds cleaved chemical protein-based reductants, respectively, integral maintaining activity, stability, substrate scope enzyme. residue oxidation an emerging regulatory strategy controlling hydrolysis pigments.

Language: Английский

Citations

1