Structural evidence for substrate‐induced synergism and half‐sites reactivity in biotin carboxylase

Abstract

Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or ''flip-flop'' their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF 2 P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC.

The enzymes of biotin dependent CO2 metabolism: What structures reveal about their reaction mechanisms

“…Further details concerning the active site pocket are presented below. This ''closed'' conformation with bound nucleotide has been subsequently observed in other biotin carboxylases 18,19 as well as in the biotin carboxylase domains of the pyruvate carboxylases from Staphylococcus aureus and Rhizobium etli cfn42. [20][21][22] One question that arises is whether the Bdomains of the biotin carboxylase dimer are completely splayed open in the absence of nucleotides.…”

“…[17][18][19] From these studies, several key interactions between the protein and the nucleotides have been identified, many of which have been confirmed by site-directed mutagenesis. Figure 3(b) summarizes the most important of these interactions.…”

“…This structure was determined in the presence of the nonhydrolyzable ATP analog AMPPNP. 18 As mentioned previously, one of the metal ions binds to the enzyme as the metal-nucleotide chelate. It is not possible to determine which magnesium ion in Figure 6(a) represents the ''metal'' in the metal-nucleotide chelate.…”

Crystal structure of the essential biotin‐dependent carboxylase AccA3 from Mycobacterium tuberculosis

“…However, the dimer interaction architecture is conserved between AccA3 and previously determined structures of biotin‐dependent carboxylases 37, 38, 39, 40. Among these there are also examples where this interaction is predicted to be stable in solution 38. Together, this suggests that the observed dimer interface is not solely a result of crystal packing but biochemically relevant.…”

“…Interestingly, the PISA analysis of the dimer interface does not suggest the formation of stable quaternary structures in solution. However, the dimer interaction architecture is conserved between AccA3 and previously determined structures of biotin‐dependent carboxylases 37, 38, 39, 40. Among these there are also examples where this interaction is predicted to be stable in solution 38.…”