Ironing out Their Differences: Dissecting the Structural Determinants of a Phenylalanine Aminomutase and Ammonia Lyase

Abstract

Deciphering the structural features that functionally separate ammonia lyases from aminomutases is of interest because it may allow for the engineering of more efficient aminomutases for the synthesis of unnatural amino acids (e.g., β-amino acids). However, this has proved to be a major challenge that involves understanding the factors that influence their activity and regioselectivity differences. Herein, we report evidence of a structural determinant that dictates the activity differences between a phenylalanine ammonia lyase (PAL) and aminomutase (PAM). An inner loop region that closes the active sites of both PAM and PAL was mutated within PAM (PAM residues 77-97) in a stepwise approach to study the effects when the equivalent residue(s) found in the PAL loop were introduced into the PAM loop. Almost all of the single loop mutations triggered a lyase phenotype in PAM. Experimental and computational evidence suggest that the induced lyase features result from inner loop mobility enhancements, which are possibly caused by a 310-helix cluster, flanking α-helices, and hydrophobic interactions. These findings pinpoint the inner loop as a structural determinant of the lyase and mutase activities of PAM.

Biochemical and Structural Analysis of Substrate Specificity of a Phenylalanine Ammonia-Lyase

“…The lattice packing of SbPAL1 was established through a homodimer as an asymmetric unit, which, in turn, formed a tight interaction with a neighboring homodimer in a 2-fold symmetry manner, indicating its homotetrameric nature with a pseudo D 2 symmetry ( Fig. 1A) as similar to other related ammonia-lyases and ammonia-transferases (Calabrese et al, 2004;Ritter and Schulz, 2004;Wang et al, 2005Wang et al, , 2008Louie et al, 2006;Moffitt et al, 2007;Heberling et al, 2015). In order to investigate the plausible oligomeric state of SbPAL1 in solution, the PISA software package (Krissinel and Henrick, 2007) was applied to evaluate interactions between neighboring molecules in crystal lattices for predicting biologically relevant oligomeric states.…”

“…On the other hand, TAL, HAL, and prokaryotic PAL/PAM lack the shielding domain and contain a longer lid-loop instead. Among those, the inner lid-loop of PcPAL was significantly different from the others and was referred to as an open active-site form (Louie et al, 2006;Heberling et al, 2015). However, due to its poor fitting into electron density, the conformation of the inner lid-loop in the deposited PcPAL structure is uncertain.…”

The cyclochlorotine mycotoxin is produced by the nonribosomal peptide synthetase CctN in Talaromyces islandicus (‘Penicillium islandicum’)

“…However, minor structural modifications could turn the lyase activity into a phenylalanine aminomutase (PAM) activity. The rigidity of an active‐site inner loop is discussed to be crucial for forcing the MIO‐amine adduct in the active site to promote re‐addition of the amino group onto cinnamic acid to form βPhe as the final product (Heberling et al ., ). In this model, the inner loop enables the chemically challenging 2,3‐amine shift when closed and causes a lyase reaction when open.…”

“…S37) which, in accordance with the findings of Heberling et al . (), may prevent the exposure to solvent and hence promote rigidity to the loop. Thus, we suggest that CctP rather acts as a PAM and provides the uncommon building block βPhe for cyclochlorotine biosynthesis.…”

Discovery and Investigation of Mutase-like Activity in a Phenylalanine Ammonia Lyase from Anabaena variabilis

“…After initial computational modelling of the complex dynamics of loop opening in TwPAM, mutations were introduced to remove any molecular constraints on this process, of which one, TwPAM-R92S, was effective [8]. In a subsequent study, additional dynamics simulations combined with loop hydrophobicity studies and comparison to a related ammonia lyase were used to create more variants with increased amination rates [9]. Due to the complexity of studying protein dynamics with a view to enzyme redesign, it is desirable to have a starting point where inner active site loop properties are already suited to amination reactions.…”