300-Fold increase in production of the Zn2+-dependent dechlorinase TrzN in soluble form via apoenzyme stabilization

Abstract

Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.

FIG 1

SDS-PAGE showing the increase in expression of soluble TrzN by laboratory evolution. The molecular mass marker is shown in lane M. Lanes 1 to 4 show the increasing cellular concentrations of expression of soluble TrzN in rounds 1 (lane 2; A159V), 2 (lane 3; A159V/L131P), and 3 (lane 4; A159V/L131P/D38N) compared with the native TrzN (lane 1).

FIG 2

Effects of the mutations in holo-TrzN-G3 (green) (A, B, and C) and apo-TrzN-G3 (cyan) (D, E, and F). (A and D) The structure of native holo-TrzN is shown in gray for comparison. The D38N mutation results in little conformational change in the holo-TrzN-G3 structure, while an alternative rotamer is adopted in apo-TrzN-G3. (B and E) The L131P mutation results in an increase in the volume of the active-site binding pocket but no other backbone changes in holo-TrzN-G3, whereas in apo-TrzN-G3, the proline residue stabilizes a tight turn in the alternative backbone conformation. (C and F) The A159V mutation results in a slight increase in an already very small cavity in native holo-TrzN, whereas in the rearranged structure of apo-TrzN-G3, a much larger cavity is formed, which is filled by the A159V mutation.

FIG 3

Comparison between apo-TrzN-G3 and holo-TrzN-G3. (a) Two loops undergo conformational change as a result of metal binding, with loop 2 (H130-Y141) in apo-TrzN-G3 (red) moving out of the active site in holo-TrzN-G3 (green). Loop 3 (S161-P180) undergoes a smaller change from apo-TrzN-G3 (magenta) to holo-TrzN-G3 (blue), which results in opening of the gorge to the active site. (b) Density from a single protein crystal structure in which the Zn²⁺ site is occupied at only 50%. The 2mf_o-Df_c, αcalc map (blue) is contoured at 1σ, with the holo-TrzN-like chain model colored cyan (50% occupancy); the mf_o-Df_c, αcalc map (green) is contoured at 2.5σ, demonstrating that the apo-TrzN-like chain (green; 50% occupancy) is also present in this structure. The second subunit of the dimer (subunit A) had 100% metal ion occupancy and no detectable density corresponding to apo-TrzN.

FIG 4

Effects of stabilizing mutations in holo-PTE and apo-PTE. (a) The A80V mutation (green) results in almost no conformational change in the holo-PTE structure (gray), only adding two methyl groups at the enzyme surface. (b) In contrast, in apo-PTE, the A80V mutation (cyan) is predicted to result in the adjacent leucine (L112) adopting a new rotamer, in which one of its methyl side chains fills a hydrophobic cavity below, which explains its predicted stabilizing effect on apo-PTE and increase in expression. (c) The R311S mutation in holo-PTE (cyan/yellow) results in the loss of a charged group on the protein surface. (d) In contrast, in apo-PTE, structural rearrangement of the apoeznyme results in R311 filling a cavity between F306 and M314. The R311S mutation allows F306 and M314 to collapse inward, filling the cavity and providing a stabilizing effect.