Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia lyase

Abstract

We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.

Figure 1

Activity profiles of A. variabilis PAL double mutant and R. toruloides PAL at different pH conditions. (a) pH stability of A. variabilis PAL double mutant and R. toruloides PAL. (b) Optimal pH profiles of A. variabilis PAL and R. toruloides PAL. All data are mean value from three parallel measurements.

Figure 2

Superimposed monomer structures of wild-type A. variabilis PAL (light brown) and the A. variabilis PAL C503S/C563S double mutant (blue). Highlighted are the two loops that are observed in an ordered conformation only in the A. variabilis PAL double mutant structure.

Figure 3

Solvent accessible surface of the A. variabilis PAL double mutant around the active site showing the inner active site loop 74–96 (shown in green colored ribbon and surface representation) acting as a door to the cavity. MIO is shown in red, Tyr78 in green, and a docked Phe molecule in magenta. Analysis with CastP shows that the active site cavity in this structure does not have a solvent accessible opening.

Figure 4

Superposition of residues in published PAL active sites (PDB code 1Y2M, 1W27, 1T6J, 1T6P, 2NYN and 2NYF) with atoms within 5.0 Å of a Phe substrate molecule docked using AutoDock shows a highly conserved active site. Residues shown in red are from the active site loop 74–96 of the A. variabilis PAL double mutant.

Figure 5

Percentage tCA bound to the A. variabilis PAL double mutant as a function of L-Phe loaded. The indicated concentration of Phe was incubated in the presence of 13 µM A. variabilis PAL double mutant for a sufficient period of time to convert all substrate to product. Samples were then desalted to remove excess L-Phe and unbound tCA and injected on a C4-RP column for analysis. Results appear to show that two PAL active sites remain occupied with the product tCA which is not easily dislodged.

Figure 6

Close-up view of active site residues that have direct interaction with the MIO and modeled tCA in the A. variabilis PAL double mutant monomer B, the only monomer that has clear electron density for the phenyl ring of tCA. Extra electron density are also observed in the other monomers, but none could be modeled as a ring. The electron density map (2F_o-F_c) is contoured at 1σ level

Figure 7

Loops from three different monomers located around each active site of A. variabilis PAL double mutant. (a) The bottom view of A. variabilis PAL with loop 74–96 (monomer A, green), 436–458 (monomer A, green), 291–311 (monomer B, orange) and 394–419 (monomer C, magenta) are highlighted. (b) Networking among residues from three monomers in each active site with MIO (red) and docked Phe (yellow).

Figure 8

Helix-loop conformational switch of aromatic ammonia lyases. (a) Superimposed monomer A, B, C and D of R. toruloides PAL (1Y2M) shows helix-loop switch in helix 88–108 region in monomer B. Monomer B: magenta; monomer A: pale cyan; monomer C: bright orange; monomer D: chartreuse. (b) Superimposed monomers of wild-type A. variabilis PAL (sky blue) and its double mutant (orange) presents disordered and well-ordered conformations of residues 75–91 region. (c) Three conformational forms of the active site loop 74–91 in A. variabilis PAL (loop-in position, orange) and its corresponding region in P. crispum PAL (loop-out position, slate) and R. toruloides PAL (disordered, cyan). In all figures, MIO, Tyr78 in A. variabilis PAL and Tyr110 in P. crispum PAL are shown in sticks.

Figure 9

Structural alignment of R. toruloides PAL (blue) and the A. variabilis PAL double mutant (salmon) monomer. (a) Rigid alignment. The C-terminal residues 498–563 in the A. variabilis PAL double mutant are aligned with C-terminal residues 652–716 in R. toruloides PAL (b) Flexible alignment. The C-terminal region of residues 498–563 in the A. variabilis PAL double mutant makes a twist and is overlapped with the region of residues 546–612, which is a part of the insertion domain in R. toruloides PAL.