Mechanistic Studies on the 8‐Amino‐7‐Oxopelargonate Synthase, a Pyridoxal‐5′‐Phosphate‐Dependent Enzyme Involved in Biotin Biosynthesis

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Abstract
The reaction mechanism of 8‐amino‐7‐oxopelargonate (8‐amino‐7‐oxononoate) synthase from Bacillus sphaericus, an enzyme dependent on pyridoxal 5′‐phosphate (pyridoxal‐P), which catalyzes the condensation of l‐alanine with pimeloyl‐CoA, the second step of biotin biosynthesis, has been studied. To facilitate mechanistic studies, an improved over‐expression system in Escherichia coli, and a new continuous spectrophotometric assay for 8‐amino‐7‐oxopelargonate synthase were designed. In order to discriminate between the two plausible basic mechanisms that can be put forth for this enzyme, that is: (a) formation of the pyridoxal‐P ‐stabilized carbanion by abstraction of the C2‐H proton of the alanine‐pyridoxal‐P aldimine, followed by acylation and decarboxylation, and (b) formation of the carbanion by decarboxylation followed by acylation, the fate of the C2‐H proton of alanine during the course of the reaction has been examined using 1H NMR. Spectra of the 8‐amino‐7‐oxopelargonate formed using either l‐[2‐2H] alanine in H2O or l‐alanine in D2O, showed that the C2‐H proton of alanine is lost during the reaction and that the C8‐H proton of 8‐amino‐7‐oxopelargonate is derived from the solvent, a result that is only consistent with mechanism (a). Futhermore 8‐amino‐7‐oxopelargonate synthase catalyzes, in the absence of pimeloyl‐CoA, the stereospecific exchange, with retention of configuration, of the C2‐H proton of l‐alanine with the solvent protons. Similarly, 8‐amino‐7‐oxopelargonate synthase catalyzes the exchange of the C8‐H proton of 8‐amino‐7‐oxopelargonate. In addition to these exchange reactions, 8‐amino‐7‐oxopelargonate synthase catalyzes an abortive transamination yielding an inactive pyridoxamine 5′‐phosphate (pyridoxamine‐P) form of 8‐amino‐7‐oxopelargonate synthase and pyruvate. Kinetic analysis gave a rate constant of kexch= 1.8 min−1 for the exchange reaction which is 10 times lower than the catalytic constant and a rate constant of ktrans= 0.11 h−1 for the transamination. Finally deuterium kinetic isotope effects (KIE) were measured at position 2 of L‐alanine (DV= 1.3) and in D2O (D2OV= 4.0). The magnitudes of the KIE are consistent with a partially rate‐limiting abstraction of the C2‐H proton of alanine and a partially rate‐limiting reprotonation step. Taken together, all these results show that 8‐amino‐7‐oxopelargonate synthase utilizes mechanism (a). 8‐Amino‐7‐oxopelargonate synthase and 5 aminolevulinate synthase, which has also been shown to use mechanism (a), belong to a class of pyridoxal‐P ‐dependent enzymes that catalyze the formation of α‐oxoamines. Based on the fact that all these α‐oxoamine synthases share strong sequence similarities, we postulate that they also share the same reaction mechanism.