Unravelling the Mechanism of Dihydrodipicolinate Synthase: are the Essential Active Site Residues Really Essential?

Abstract

Dihydrodipicolinate synthase (DHDPS, an important antibiotic target) is the enzyme that catalyses the first committed step in the lysine biosynthetic pathway, which involves the condensation reaction between (S)-aspartate β-semialdehyde ((S)-ASA) and pyruvate via a ping-pong mechanism, and is feedback inhibited by lysine. The major hallmark of this reaction is the formation of a Schiff base intermediate between pyruvate and the active site residue lysine 161. Surprisingly, this had never been confirmed using site-directed mutagenesis. To investigate the necessity of this residue, two site-directed mutants were generated: DHDPS-K161A and DHDPS-K161R. They were then over-expressed, purified and characterised by steady-state kinetics, circular dichroism (CD)spectroscopy, differential scanning fluorimetry (DSF),isothermal titration calorimetry (ITC), sodium borohydride reduction and X-ray crystallography. Unexpectedly, the mutant enzymes were still catalytically active, albeit with substantially impaired catalytic competency, underscoring the functional plasticity of enzyme active sites. These results are in contrast to findings in the structurally-related enzyme, N-acetyl neuraminate lyase (NAL) and hint at evolutionary relationships in the class I aldolase family.