Ironing out their differences: dissecting the structural determinants of a phenylalanine aminomutase and ammonia lyase.

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., ß-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.

Structural investigations into the stereochemistry and activity of a phenylalanine-2,3-aminomutase from Taxus chinensis.

Phenylalanine-2,3-aminomutase (PAM) from Taxus chinensis, a 4-methylidene-imidazole-5-one (MIO)-dependent enzyme, catalyzes the reversible conversion of (S)-α-phenylalanine into (R)-ß-phenylalanine via trans-cinnamic acid. The enzyme also catalyzes the direct addition of ammonia to trans-cinnamic acid, a reaction that can be used for the preparation of ß-amino acids, which occur as frequent constituents of bioactive compounds. Different hypotheses have been formulated to explain the stereochemistry of the PAM-catalyzed reaction, but structural evidence for these hypotheses is lacking. Furthermore, it remains unclear how the PAM MIO group is formed from the three-amino acid (A-S-G) sequence motif. For these reasons, we elucidated PAM three-dimensional (3D) structures with a bound (R)-ß-phenylalanine analogue and with bound trans-cinnamic acid. In addition, 3D structures of the (inactive) Y322A and N231A mutants of PAM were elucidated, which were found to be MIO-less. We conclude that the stereochemistry of the PAM-catalyzed reaction originates from the enzyme's ability to bind trans-cinnamic acid in two different orientations, with either the si,si face or the re,re face directed toward the MIO group, as evidenced by two distinct carboxylate binding modes. The results also suggest that the N231 side chain promotes MIO group formation by increasing the nucleophilicity of the G177 N atom through acidification of the amide proton.

Biochemical properties and crystal structure of a ß-phenylalanine aminotransferase from Variovorax paradoxus.

By selective enrichment, we isolated a bacterium that can use ß-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg(-1) for (S)-ß-phenylalanine at 30°C and 33 U mg(-1) at the optimum temperature of 55°C. The ß-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted ß-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-ß-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic ß-amino acids to yield (R)-ß-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the ß-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic ß-amino acid-converting aminotransferases may be identified.

Structural determinants of the ß-selectivity of a bacterial aminotransferase.

Chiral ß-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure ß-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-ß-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a ß-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A ß-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both ß- and α-amino acids.

Mechanism-inspired engineering of phenylalanine aminomutase for enhanced ß-regioselective asymmetric amination of cinnamates.

Turn to switch: A mutant of phenylalanine aminomutase was engineered that can catalyze the regioselective amination of cinnamate derivatives (see scheme, red) to, for example, ß-amino acids. This regioselectivity, along with the X-ray crystal structures, suggests two distinct carboxylate binding modes differentiated by C(ß)-C(ipso) bond rotation, which determines if ß- (see scheme) or α-addition takes place.