A novel genetic selection system for PLP-dependent threonine aldolases

Abstract

Threonine aldolases are versatile pyridoxal-5′-phosphate (PLP)-dependent enzymes key to glycine, serine and threonine metabolism. Because they catalyze the reversible addition of glycine to an aldehyde to give β-hydroxy-α-amino acids, they are also attractive as biotechnological catalysts for the diastereoselective synthesis of many pharmaceutically useful compounds. To study and evolve such enzymes, we have developed a simple selection system based on the simultaneous inactivation of four genes involved in glycine biosynthesis in Escherichia coli. Glycine prototrophy in the deletion strain is restored by expression of a gene encoding an aldolase that converts β-hydroxy-α-amino acids, provided in the medium, to glycine and the corresponding aldehyde. Combinatorial mutagenesis and selection experiments with a previously uncharacterized l-threonine aldolase from Caulobacter crescentus CB15 (Cc-LTA) illustrate the power of this system. The codons for four active site residues, His91, Asp95, Glu96, and Asp176, were simultaneously randomized and active variants selected. The results show that only His91, which π-stacks against the PLP cofactor and probably serves as the catalytic base in the carbon-carbon bond cleavage step, is absolutely required for aldolase activity. In contrast, Asp176, one of the most conserved residues in this enzyme superfamily, can be replaced conservatively by glutamate, albeit with a >5000-fold decrease in efficiency. Though neither Asp95 nor Glu96 is catalytically essential, they appear to modulate substrate binding and His91 activity, respectively. The broad dynamic range of this novel selection system should make it useful for mechanistic investigations and directed evolution of many natural and artificial aldolases.