7,8‐Diaminoperlargonic acid aminotransferase from Mycobacterium tuberculosis , a potential therapeutic target

Diaminopelargonic acid aminotransferase (DAPA AT), which is involved in biotin biosynthesis, catalyzes the transamination of 8‐amino‐7‐oxononanoic acid (KAPA) using S ‐adenosyl‐ l ‐methionine (AdoMet) as amino donor. Mycobacterium tuberculosis DAPA AT, a potential therapeutic target, has been overproduced in Escherichia coli and purified to homogeneity using a single efficient step on a nickel‐affinity column. The enzyme shows an electronic absorption spectrum typical of pyridoxal 5′‐phosphate‐dependent enzymes and behaves as a homotetramer in solution. The pH profile of the activity at saturation shows a single ionization group with a p K a of 8.0, which was attributed to the active‐site lysine residue. The enzyme shows a Ping Pong Bi Bi kinetic mechanism with strong substrate inhibition with the following parameters: K mAdoMet  = 0.78 ± 0.20 m m , K mKAPA  = 3.8 ± 1.0 µ m , k cat  = 1.0 ± 0.2 min −1 , K iKAPA  = 14 ± 2 µ m . Amiclenomycin and a new analogue, 4‐(4 c ‐aminocyclohexa‐2,5‐dien‐1 r ‐yl)propanol (referred to as compound  1 ), were shown to be suicide substrates of this enzyme, with the following inactivation parameters: K i  = 12 ± 2 µ m , k inact  = 0.35 ± 0.05 min −1 , and K i  = 20 ± 2 µ m , k inact  = 0.56 ± 0.05 min −1 , for amiclenomycin and compound  1 , respectively. The inactivation was irreversible, and the partition ratios were 1.0 and 1.1 for amiclenomycin and compound  1 , respectively, which make these inactivators particularly efficient. compound  1 (100 µg·mL −1 ) completely inhibited the growth of an E. coli C268bioA mutant strain transformed with a plasmid expressing the M. tuberculosis bioA gene, coding for DAPA AT. Reversal of the antibiotic effect was observed on the addition of biotin or DAPA. Thus, compound  1 specifically targets DAPA AT in vivo .