Controlling Enzymatic Activity by Modulating the Oligomerization State via Chemical Rescue and Optical Control

Kropp, Cosimo; Bruckmann, Astrid; Babinger, Patrick

doi:10.1002/cbic.202100490

Cited by 5 publications

(2 citation statements)

References 37 publications

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“…Interestingly, it has been also reported that oligomerization of enzymes modulates their activities ( Kropp et al, 2022 ). We showed here that CypB dimer formation was fully required to complement the defective phenotype of Brucella Δc ypAB mutant in stress survival, intracellular adaptation, and virulence in mice but not required for the in vitro PPIase activity.…”

Section: Discussionmentioning

confidence: 99%

Biochemical and functional characterization of Brucella abortus cyclophilins: So similar, yet so different

Muruaga

Briones²,

Roset³

2022

Front. Microbiol.

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Brucella spp. are the etiological agent of animal and human brucellosis. We have reported previously that cyclophilins of Brucella (CypA and CypB) are upregulated within the intraphagosomal replicative niche and required for stress adaptation and host intracellular survival and virulence. Here, we characterize B. abortus cyclophilins, CypA, and CypB from a biochemical standpoint by studying their PPIase activity, chaperone activity, and oligomer formation. Even though CypA and CypB are very similar in sequence and share identical chaperone and PPIase activities, we were able to identify outstanding differential features between them. A series of differential peptide loops were predicted when comparing CypA and CypB, differences that might explain why specific antibodies (anti-CypA or anti-CypB) were able to discriminate between both cyclophilins without cross-reactivity. In addition, we identified the presence of critical amino acids in CypB, such as the Trp134 which is responsible for the cyclosporin A inhibition, and the Cys128 that leads to CypB homodimer formation by establishing a disulfide bond. Here, we demonstrated that CypB dimer formation was fully required for stress adaptation, survival within HeLa cells, and mouse infection in B. abortus. The presence of Trp134 and the Cys128 in CypB, which are not present in CypA, suggested that two different kinds of cyclophilins have evolved in Brucella, one with eukaryotic features (CypB), another (CypA) with similar features to Gram-negative cyclophilins.

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Section: Discussionmentioning

confidence: 99%

Biochemical and functional characterization of Brucella abortus cyclophilins: So similar, yet so different

Muruaga

Briones²,

Roset³

2022

Front. Microbiol.

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“…However, this approach suffers from low selectivity and versatility. These problems have been overcome by genetic code expansion, which uses the reprogramming of a stop codon to incorporate ncAAs at practically any desired position of a protein of interest. − In this respect, photoxenoprotein engineering has enabled photocontrol of protein functions including modulation of enzyme activity and allostery, protein binding affinities, protein localization, and protein assembly and multimerization. − …”

Section: Introductionmentioning

confidence: 99%

Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase

et al. 2022

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The artificial regulation of enzymatic activity by light is an important goal of synthetic biology that can be achieved by the incorporation of light-responsive noncanonical amino acids via genetic code expansion. Here, we apply this concept to anthranilate synthase from Salmonella typhimurium (stTrpE). This enzyme catalyzes the first step of tryptophan biosynthesis, and its activity is feedback-inhibited by the binding of the end-product of the pathway to an allosteric site. To put this feedback inhibition of stTrpE by tryptophan under the control of light, we individually replaced 15 different amino acid residues with the photosensitive noncanonical amino acid o-nitrobenzyl-O-tyrosine (ONBY). ONBY contains a sterically demanding caging group that was meant to cover the allosteric site. Steady-state enzyme kinetics showed that the negative effect of tryptophan on the catalytic activity of the two variants stTrpE-K50ONBY and stTrpE-Y455ONBY was diminished compared to the wild-type enzyme by 1 to 2 orders of magnitude. Upon light-induced decaging of ONBY to the less space-consuming tyrosine residue, tryptophan binding to the allosteric site was restored and catalytic activity was inhibited almost as efficiently as observed for wild-type stTrpE. Based on these results, direct photocontrol of feedback inhibition of stTrpE-K50ONBY and stTrpE-Y455ONBY could be achieved by irradiation during the reaction. Molecular modeling studies allowed us to rationalize the observed functional conversion from the noninhibited caged to the tryptophan-inhibited decaged states. Our study shows that feedback inhibition, which is an important mechanism to regulate key metabolic enzymes, can be efficiently controlled by the purposeful use of light-responsive noncanonical amino acids.

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Controlling Enzymatic Activity by Modulating the Oligomerization State via Chemical Rescue and Optical Control

2021

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Selective switching of enzymatic activity has been a longstanding goal in synthetic biology. Drastic changes in activity upon mutational manipulation of the oligomerization state of enzymes have frequently been reported in the literature, but scarcely exploited for switching. Using geranylgeranylglyceryl phosphate synthase as a model, we demonstrate that catalytic activity can be efficiently controlled by exogenous modulation of the association state. We introduced a lysine-to-cysteine mutation, leading to the breakdown of the active hexamer into dimers with impaired catalytic efficiency. Addition of bromo-ethylamine chemically rescued the enzyme by restoring hexamerization and activity. As an alternative method, we incorporated the photosensitive unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) into the hexamerization interface. This again led to inactive dimers, but the hexameric state and activity could be recovered by UV-light induced cleavage of ONBY. For both approaches, we obtained switching factors greater than 350-fold, which compares favorably with previously reported activity changes that were caused by sitedirected mutagenesis.

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Controlling Enzymatic Activity by Modulating the Oligomerization State via Chemical Rescue and Optical Control

Cited by 5 publications

References 37 publications

Biochemical and functional characterization of Brucella abortus cyclophilins: So similar, yet so different

Biochemical and functional characterization of Brucella abortus cyclophilins: So similar, yet so different

Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase

Controlling Enzymatic Activity by Modulating the Oligomerization State via Chemical Rescue and Optical Control

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