SpyRing interrogation: analyzing how enzyme resilience can be achieved with phytase and distinct cyclization chemistries

Abstract

Enzymes catalyze reactions with exceptional selectivity and rate acceleration but are often limited by instability. Towards a generic route to thermo-resilience, we established the SpyRing approach, cyclizing enzymes by sandwiching between SpyTag and SpyCatcher (peptide and protein partners which lock together via a spontaneous isopeptide bond). Here we first investigated the basis for this resilience, comparing alternative reactive peptide/protein pairs we engineered from Gram-positive bacteria. Both SnoopRing and PilinRing cyclization gave dramatic enzyme resilience, but SpyRing cyclization was the best. Differential scanning calorimetry for each ring showed that cyclization did not inhibit unfolding of the inserted β-lactamase. Cyclization conferred resilience even at 100 °C, where the cyclizing domains themselves were unfolded. Phytases hydrolyze phytic acid and improve dietary absorption of phosphate and essential metal ions, important for agriculture and with potential against human malnutrition. SpyRing phytase (PhyC) resisted aggregation and retained catalytic activity even following heating at 100 °C. In addition, SpyRing cyclization made it possible to purify phytase simply by heating the cell lysate, to drive aggregation of non-cyclized proteins. Cyclization via domains forming spontaneous isopeptide bonds is a general strategy to generate resilient enzymes and may extend the range of conditions for isolation and application of enzymes.

Figure 1. Enzyme cyclization using alternative isopeptide-forming domains.

(A) Schematic of SnoopRing and PilinRing cyclization of β-lactamase (BLA). Proteins are shown in cartoon format, with reacting residues shown in green in stick format. (B) SDS-PAGE analysis of SpyRing, SnoopRing and PilinRing cyclization. Each purified protein is shown alongside the non-reactive control (DA or KA), after staining with Coomassie.

Figure 2. SnoopRing and PilinRing cyclization conferred thermal resilience.

(A) Cyclization decreased heat-induced aggregation. BLA, Pilin-C-BLA-Isopeptag and SnoopTag-BLA-SnoopCatcher were heated at the indicated temperature for 10 min, centrifuged, and the supernatant analyzed by SDS-PAGE with Coomassie staining. C is control without incubation. (B) Plot of heat-induced aggregation, from quantifying SDS-PAGE. (C) Cyclization enhanced the catalytic activity following heating. BLA, SnoopTag-BLA-SnoopCatcher, Pilin-C-BLA-Isopeptag and SpyTag-BLA-SpyCatcher were incubated for 10 min at the indicated temperature, before returning to RT and then running a nitrocefin colorimetric activity assay. The initial rates of reaction were calculated using the linear portion of the enzyme curves. Data were normalized using the initial rate of reaction at 25 °C as 100% recovered activity. (All are mean of triplicate ± 1 s.d.; some error bars are too small to be visible.)

Figure 3. DSC comparison of SpyRing, SnoopRing and PilinRing BLA.

(A) DSC showing the melting profiles of BLA overlaid with SpyCatcher + SpyTag peptide, SnoopCatcher + SnoopTag peptide, and Spy0128, giving the specific heat capacity (Cp) of each protein scanned from 20–100 °C at 1 °C/min. (B) DSC showing the melting profile of SpyTag-BLA-SpyCatcher overlaid with SnoopTag (KA)-BLA-SnoopCatcher or Pilin-C (KA)-BLA-Isopeptag, analyzed as in (A). (C) Tm values for peaks observed in (B).

Figure 4. SpyRing cyclization of phytase increased the thermal resilience.

(A) Crystal structure of PhyC phytase from Bacillus subtilis in cartoon format, with the substrate analog myo-inositol hexasulfate in stick format (based on PDB 3AMR). Spheres represent calcium ions and the distance between termini is marked. (B) Cyclization caused a gel shift. SpyTag-PhyC-SpyCatcher (ST), SpyTag DA-PhyC-SpyCatcher (DA) and PhyC (WT) were boiled in SDS-loading buffer and analyzed by SDS-PAGE with Coomassie staining. (C) SpyRing cyclization improved aggregation-resistance. SpyTag-PhyC-SpyCatcher or PhyC were heated at the indicated temperature for 10 min, centrifuged and the supernatant analyzed by SDS-PAGE with Coomassie staining. C is control without incubation. Data from triplicate measurements were then plotted. (D) SpyRing cyclization improved the recovered activity after heating. SpyTag-PhyC-SpyCatcher or PhyC were incubated for 10 min at the indicated temperature, before returning to RT and then running a colorimetric activity assay for phosphate release from phytic acid. (All are mean of triplicate ± 1 s.d.; some error bars are too small to be visible.)

Figure 5. Analysis of SpyRing phytase by DSC.

DSC of SpyTag (DA)-PhyC-SpyCatcher and PhyC. DSC gave the specific heat capacity (Cp) of each enzyme, scanning from 20–110 °C at 1 °C/min.

Figure 6. SpyRing cyclization allowed enzyme purification just by heating.

(A) Heat-based purification of phytase. E. coli lysate expressing PhyC or SpyTag-PhyC-SpyCatcher was heated for 10 min at the indicated temperature, centrifuged, and the supernatant analyzed by SDS-PAGE with Coomassie staining. C is control without incubation. Mw are the molecular weight markers. (B) Comparing purity between heat-purified and affinity-purified phytase. SpyTag-PhyC-SpyCatcher either affinity-purified by Ni-NTA or heat-purified (normalized to give an equal intensity of the SpyTag-PhyC-SpyCatcher band) was boiled in SDS-loading buffer and analyzed by SDS-PAGE with Coomassie staining. (C) Heat purified phytase was still active. Catalytic activity of SpyTag-PhyC-SpyCatcher purified by either affinity chromatography or by heat purification (normalized by densitometry for an equal intensity of the SpyTag-PhyC-SpyCatcher band) was tested by a colorimetric activity assay for phosphate release from phytic acid. (All are mean of triplicate ± 1 s.d.; some error bars are too small to be visible.)