Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) 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. In an attempt 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) and X-ray crystallography. Wild-type DHDPS was also over-expressed, purified and characterised in order to provide baseline data for comparison. Unexpectedly, the mutant enzymes were still catalytically active, despite a significant decrease in activity. The kcat values for DHDPS-K161A and DHDPS-K161R were 0.06±0.02 s⁻¹ and 0.16±0.06 s⁻¹ respectively, compared to 45±3 s⁻¹ for the wild-type enzyme. Another interesting finding was a shift in the mechanism, from ping-pong to ternary-complex. This implies that the strict order of reaction sequences observed in the wild-type enzyme was relaxed in the mutant enzymes. The Km values with respect to pyruvate increased by only 3-fold for both enzymes (0.45±0.04 mM for DHDPS-K161A and 0.57±0.06 mM for DHDPS-K161R, compared to 0.15±0.01 mM for the wild-type DHDPS), indicating a subtle change in pyruvate binding affinity; while the KmASA value remained the same for DHDPS-K161R (0.12±0.01 mM) and increased by 2-fold for DHDPS-K161A (0.23±0.02 mM). Both enzymes behaved in an entirely analogous fashion to the wild-type enzyme in regards to lysine inhibition, demonstrating the lack of involvement of lysine 161 in the regulatory mechanism. Treatment with the reducing agent sodium borohydride provided evidence that a Schiff base is not formed when pyruvate binds to either one of the mutant enzymes. DSF showed that the melting temperature for DHDPS-K161A and DHDPS-K161R upon addition of pyruvate increased to a lesser extent than for the wild-type enzyme, suggesting that stabilisation of DHDPS by pyruvate is largely dependent on Schiff base formation. CD spectroscopy showed that the mutant enzymes had identical secondary structures in solution to the wild-type enzyme. The crystal structures of DHDPS-K161A and DHDPS-K161R were also solved to resolutions of 2.0 and 2.4 Å respectively. They showed no major rearrangement within their active sites, indicating that their attenuated catalytic activities were due to the removal of lysine 161, as opposed to gross structural alterations induced by the mutations. The crystal structure of the mutant enzymes were also solved with pyruvate bound and they showed that both mutant enzymes still had a well defined binding pocket for pyruvate that is not dependent of lysine 161. ITC experiments demonstrated that in the absence of Schiff base formation, entropic contributions may be responsible for the observed enzymatically accelerated reaction of the two DHDPS substrates, since no discernible heat was produced upon binding of pyruvate to the mutant enzymes. Based on these results, it can be concluded that although lysine 161 is important in the DHDPS-catalysed reaction, it is not absolutely essential. The current proposed mechanism, where the tautomerisation of the Schiff base to an enamine species creates the nucleophile necessary for attack of (S)-ASA, had to be reconsidered and expanded. We propose that the enol form of pyruvate, which exists in equilibrium with its keto tautomer, can fulfill the role of the enamine species. NMR studies using hydrogen-deuterium exchange suggest that the rate of enolisation is unchanged in DHDPS-K161A and DHDPS-K161R catalysed mechanism, ruling out the possibility that the enolisation process could be the rate-determining step. It is likely that substrate orientation and favourable charge interactions may facilitate the interaction of an active site bound pyruvate with (S)-ASA and may be the major catalytic devices operating in DHDPS-K161A and DHDPS-K161R. Removal of the Schiff base-forming residue in DHDPS could not impede catalysis completely because of the existence of this 'back-up' mechanism, in which lysine 161 plays no part. These conclusions may shed light into the mechanism and evolution of enzymatic activity of other class I aldolases and they underscore the functional plasticity of enzyme active sites.