Structural and Biochemical Characterization of the Therapeutic Anabaena variabilis Phenylalanine Ammonia Lyase

Abstract

We have recently observed promising success in a mouse model at treating the metabolic disorder phenylketonuria (PKU) with phenylalanine ammonia lyase (PAL) from R. toruloides and A. variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability and aggregation. We reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Here we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant. 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 NHS-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylideneimidazole-5-one (MIO) 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.

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

“…The high pH optimum of the reactions may be a reflection of the reaction mechanism and is generally identical for all enzymes and similar to what has been observed previously for RsTAL, SeSam8, and other homologous enzymes (18,20,21,23,27,(78)(79)(80)(81). Alkaline pH may also be used for biocatalysis, where tyrosine produced by fermentation at physiological pH can be converted to pHCA in high titers by subsequent reaction at alkaline pH in the presence of TAL-expressing E. coli cells or cell paste (82).…”

“…Several enzymes have previously been characterized both in vivo and in vitro, and the data are available from both patent and scientific literature (3,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). However, the conditions of kinetic analysis are often quite different and may not represent the extent to which the enzymes perform in vivo.…”

Preparation of Unnatural Amino Acids with Ammonia-Lyases and 2,3-Aminomutases

“…Besides their biocatalytic usefulness, MIO enzymes have medical aspects like better understanding of the genetics of HAL regulation for the treatment of histidinaemia (53) or use PAL as a therapeutic agent for the treatment of phenylketonuria (54,55).…”

Biotechnological production and applications of microbial phenylalanine ammonia lyase: a recent review

“…They presumed that the formation of the dehydroalanine residue at the catalytic centre of PAL was preceeded by an autocatalytic mechanism, rather than by exogenous chemical modification. However, this assumption is incorrect, because the co-factor of PAL has been proven to be MIO (Calabrese et al, 2004;Moffitt et al, 2007;Wang et al, 2008). Faulkner et al (1994) constructed a bi-functional promoter (pPGK::REP2).…”

“…This homotetrameric protein contains 716 residues per subunit with a molecular mass of 76.88 kDa. Recently, the three-dimensional structure of PAL from Anabaena variabilis (Moffitt et al, 2007) and Nostoc punctiforme (Wang et al, 2008) has also been described using X-ray crystallography. The PALs from A. variabilis and N. punctiforme exist as a homotetramer with 222-point symmetry.…”