Designing Allosteric Control into Enzymes by Chemical Rescue of Structure

Deckert, Katelyn; Budiardjo, S Jimmy; Brunner, Luke C; Lovell, Scott; Karanicolas, John

doi:10.1021/ja301409g

Title: Designing Allosteric Control into Enzymes by Chemical Rescue of Structure

Full Record
Other Related Research

Abstract

Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a {beta}-glycosidase enzyme ({beta}-gly) and W492G in a {beta}-glucuronidase enzyme ({beta}-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of {beta}-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of {beta}-gly W33G, which together establish the structural basis for enzymemore »« less

Authors:

Deckert, Katelyn; Budiardjo, S Jimmy; Brunner, Luke C; Lovell, Scott; Karanicolas, John ^[1]

Kansas

Publication Date:: Tue Aug 07 00:00:00 EDT 2012

Research Org.:: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Org.:: OTHERNIHNIGMS

OSTI Identifier:: 1047899

Resource Type:: Journal Article

Journal Name:: Journal of the American Chemical Society

Additional Journal Information:: Journal Volume: 134; Journal Issue: 24; Journal ID: ISSN 0002-7863

Country of Publication:: United States

Language:: ENGLISH

Subject:: 59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; CHEMISTRY; CRYSTAL STRUCTURE; ENGINEERS; ENZYMES; INACTIVATION; INDOLES; KINETICS; MORPHOLOGY; PROTEIN STRUCTURE; SENSORS; TRYPTOPHAN

Citation Formats


                    Deckert, Katelyn, Budiardjo, S Jimmy, Brunner, Luke C, Lovell, Scott, and Karanicolas, John. Designing Allosteric Control into Enzymes by Chemical Rescue of Structure.  United States: N. p., 2012. 
        Web.  doi:10.1021/ja301409g.

Copy to clipboard


                    Deckert, Katelyn, Budiardjo, S Jimmy, Brunner, Luke C, Lovell, Scott, & Karanicolas, John. Designing Allosteric Control into Enzymes by Chemical Rescue of Structure.  United States.  https://doi.org/10.1021/ja301409g

Copy to clipboard


                    Deckert, Katelyn, Budiardjo, S Jimmy, Brunner, Luke C, Lovell, Scott, and Karanicolas, John. 2012.  
        "Designing Allosteric Control into Enzymes by Chemical Rescue of Structure".  United States.  https://doi.org/10.1021/ja301409g.

Copy to clipboard


                    
@article{osti_1047899,

  title        = {Designing Allosteric Control into Enzymes by Chemical Rescue of Structure},

  author       = {Deckert, Katelyn and Budiardjo, S Jimmy and Brunner, Luke C and Lovell, Scott and Karanicolas, John},

  abstractNote = {Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a {beta}-glycosidase enzyme ({beta}-gly) and W492G in a {beta}-glucuronidase enzyme ({beta}-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of {beta}-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of {beta}-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate {beta}-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.},

  doi          = {10.1021/ja301409g},

  url          = {https://www.osti.gov/biblio/1047899},
  journal      = {Journal of the American Chemical Society},

  issn         = {0002-7863},

  number       = 24,

  volume       = 134,

  place        = {United States},

  year         = {Tue Aug 07 00:00:00 EDT 2012},

  month        = {Tue Aug 07 00:00:00 EDT 2012}

}

Copy to clipboard

Journal Article:

https://doi.org/10.1021/ja301409g

Other availability

Find in Google Scholar

Search WorldCat to find libraries that may hold this journal

Save / Share:

Export Metadata

Save to My Library

Similar records in OSTI.GOV collections:

Structural Insight into Allosteric Inhibition of Mycobacterium tuberculosis Tryptophan Synthase

Journal Article Michalska, Karolina; Wellington, Samantha; Nag, Partha; ... - FASEB Journal

The rise of antibiotic‐resistant pathogens requires redirecting drug discovery effort toward new cellular targets to provide new treatment options. Such alternative, yet unexplored opportunities exist in central metabolism, where many enzymes are only conditionally essential and thus their successful exploitation requires understanding of metabolic fluxes and pathway regulations under certain environmental conditions. In Mycobacterium tuberculosis , tryptophan biosynthesis represents one such promising pathway that could be targeted. In Mtb, the multistep L‐Trp synthesis involves several enzymes: TrpEG, TrpD, TrpC and TrpAB, which is validated as conditionally essential in vivo . The whole‐cell screening of a diversity‐oriented synthetic library identified amore »« less
https://doi.org/10.1096/fasebj.31.1_supplement.765.12
Allosteric inhibitors of Mycobacterium tuberculosis tryptophan synthase

Journal Article Michalska, Karolina; Chang, Changsoo; Maltseva, Natalia; ... - Protein Science

Global dispersion of multidrug resistant bacteria is very common and evolution of antibiotic-resistance is occurring at an alarming rate, presenting a formidable challenge for humanity. The development of new therapeuthics with novel molecular targets is urgently needed. Current drugs primarily affect protein, nucleic acid, and cell wall synthesis. Metabolic pathways, including those involved in amino acid biosynthesis, have recently sparked interest in the drug discovery community as potential reservoirs of such novel targets. Tryptophan biosynthesis, utilized by bacteria but absent in humans, represents one of the currently studied processes with a therapeutic focus. It has been shown that tryptophan synthasemore »« less
Cited by 18
https://doi.org/10.1002/pro.3825

Full Text Available
Catalytically impaired TrpA subunit of tryptophan synthase from Chlamydia trachomatis is an allosteric regulator of TrpB

Journal Article Michalska, Karolina; Wellington, Samantha; Maltseva, Natalia; ... - Protein Science

Abstract Intracellular growth and pathogenesis of Chlamydia species is controlled by the availability of tryptophan, yet the complete biosynthetic pathway for l‐ Trp is absent among members of the genus. Some representatives, however, preserve genes encoding tryptophan synthase, TrpAB – a bifunctional enzyme catalyzing the last two steps in l‐ Trp synthesis. TrpA (subunit α) converts indole‐3‐glycerol phosphate into indole and glyceraldehyde‐3‐phosphate (α reaction). The former compound is subsequently used by TrpB (subunit β) to produce l‐ Trp in the presence of l‐ Ser and a pyridoxal 5′‐phosphate cofactor (β reaction). Previous studies have indicated that in Chlamydia , TrpAmore »« less
https://doi.org/10.1002/pro.4143
Conservation of the structure and function of bacterial tryptophan synthases

Journal Article Michalska, Karolina; Gale, Jennifer; Joachimiak, Grazyna; ... - IUCrJ

Tryptophan biosynthesis is one of the most characterized processes in bacteria, in which the enzymes fromSalmonella typhimuriumandEscherichia coliserve as model systems. Tryptophan synthase (TrpAB) catalyzes the final two steps of tryptophan biosynthesis in plants, fungi and bacteria. This pyridoxal 5'-phosphate (PLP)-dependent enzyme consists of two protein chains, α (TrpA) and β (TrpB), functioning as a linear αββα heterotetrameric complex containing two TrpAB units. The reaction has a complicated, multistep mechanism resulting in the β-replacement of the hydroxyl group of L-serine with an indole moiety. Recent studies have shown that functional TrpAB is required for the survival of pathogenic bacteria inmore »« less
Cited by 11
https://doi.org/10.1107/S2052252519005955

Full Text Available
Evolution of allosteric regulation in chorismate mutases from early plants

Journal Article Kroll, Kourtney; Holland, Cynthia; Starks, Courtney; ... - Biochemical Journal

Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plantmore »« less
https://doi.org/10.1042/BCJ20170549

Similar Records