phenylserine and benzaldehyde

An inducible phenylserine aldolase (L-threo-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26), which catalyzes the cleavage of L-3-phenylserine to yield benzaldehyde and glycine, was purified to homogeneity from a crude extract of Pseudomonas putida 24-1 isolated from soil. The enzyme was a hexamer with the apparent subunit molecular mass of 38 kDa and contained 0.7 mol of pyridoxal 5' phosphate per mol of the subunit. The enzyme exhibited absorption maxima at 280 and 420 nm. The maximal activity was obtained at about pH 8.5. The enzyme acted on L-threo-3-phenylserine (Km, 1.3 mM), l-erythro-3-phenylserine (Km, 4.6 mM), l-threonine (Km, 29 mM), and L-allo-threonine (Km, 22 mM). In the reverse reaction, threo- and erythro- forms of L-3-phenylserine were produced from benzaldehyde and glycine. The optimum pH for the reverse reaction was 7.5. The structural gene coding for the phenylserine aldolase from Pseudomonas putida 24-1 was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the phenylserine aldolase gene encoded a peptide containing 357 amino acids with a calculated molecular mass of 37.4 kDa. The recombinant enzyme was purified and characterized. Site-directed mutagenesis experiments showed that replacement of K213 with Q resulted in a loss of the enzyme activity, with a disappearance of the absorption maximum at 420 nm. Thus, K213 of the enzyme probably functions as an essential catalytic residue, forming a Schiff base with pyridoxal 5'-phosphate.

Topics: Aldehyde-Lyases; Amino Acid Sequence; Base Sequence; Benzaldehydes; Binding Sites; Enzyme Induction; Escherichia coli; Glycine; Hydrogen-Ion Concentration; Molecular Sequence Data; Pseudomonas putida; Sequence Analysis, DNA; Serine; Substrate Specificity

Alanine racemase (Alr) [EC 5.1.1.1] from Geobacillus stearothermophilus is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the first committed step in bacterial cell wall biosynthesis. It is converted to an aldolase upon replacement of Tyr265, which normally serves as a catalytic base in the racemase reaction, with alanine. The Y265A mutation increases catalytic efficiency for cleavage of beta-phenylserine to benzaldehyde and glycine by 2.3 x 105 fold as compared to the wild-type racemase, while racemase activity is greatly decreased. Additional mutagenesis suggests that His166 may act as the base that initiates the retroaldol reaction. The Y265A mutant is highly stereoselective for (2R,3S)-phenylserine, a d-amino acid, and does not process its enantiomer. This preference is consistent with the expected binding mode of substrate in the modified active site and supports the proposal that naturally occurring d-threonine aldolases and alanine racemases derive from a common ancestor.

Topics: Alanine Racemase; Amino Acid Substitution; Benzaldehydes; Binding Sites; Fructose-Bisphosphate Aldolase; Glycine; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Pyridoxal Phosphate; Serine