The Three-Dimensional Structure of the Biotin Carboxylase-Biotin Carboxyl Carrier Protein Complex of E. coli Acetyl-CoA Carboxylase

Abstract

SUMMARY Acetyl-coenzyme A (acetyl-CoA) carboxylase is a biotin-dependent, multifunctional enzyme that catalyzes the regulated step in fatty acid synthesis. The Escherichia coli enzyme is composed of a homodimeric biotin carboxylase (BC), biotinylated biotin carboxyl carrier protein (BCCP), and an α2β2 heterotetrameric carboxyltransferase. This enzyme complex catalyzes two half-reactions to form malonylcoenzyme A. BC and BCCP participate in the first half-reaction, whereas carboxyltransferase and BCCP are involved in the second. Three-dimensional structures have been reported for the individual subunits; however, the structural basis for how BCCP reacts with the carboxylase or transferase is unknown. Therefore, we report here the crystal structure of E. coli BCCP complexed with BC to a resolution of 2.49 Å. The protein-protein complex shows a unique quaternary structure and two distinct interfaces for each BCCP monomer. These BCCP binding sites are unique compared to phylogenetically related biotin-dependent carboxylases and therefore provide novel targets for developing antibiotics against bacterial acetyl-CoA carboxylase.

Non-Catalytic Subunits Facilitate Quaternary Organization of Plastidic Acetyl-CoA Carboxylase

“…The structural organization of fatty acid synthases and polyketide synthases also support this supposition (Smith and Sherman, 2008;Chan and Vogel, 2010;Bukhari et al, 2014). Structural characterization of these enzymes indicate catalytic coordination is facilitated by the quaternary organization of the catalytic subsites, which have to be iteratively visited by intermediates of the overall reaction (Broussard et al, 2013;Tran et al, 2015;Wei and Tong, 2015;Hunkeler et al, 2016).…”

Search for novel targets of the PII signal transduction protein in Bacteria identifies the BCCP component of acetyl‐CoA carboxylase as a PII binding partner

“…The structure of the E. coli BC‐BCCP was solved recently, the BCCP thumb is solvent exposed within the BC‐BCCP complex (Broussard et al ., ). Strikingly, we were not able to detect interaction between native E. coli GlnK and the His‐BC:holo‐BCCP complex mobilized on a Ni 2+ magnetic resin, suggesting that holo‐BCCP cannot interact with GlnK and BC simultaneously.…”

A Family of Negative Regulators Targets the Committed Step of de Novo Fatty Acid Biosynthesis

“…However, given the similarity of BADC and BCCP with respect to primary amino acid sequence ( Figure 5A), predicted structure ( Figure 5B), and dimerization capability (Figure 4), the parsimonious interpretation of these data is that BADC proteins inhibit hetACCase activity by competing with BCCP for binding to other hetACCase subunits. The crystal structure of the E. coli BC/BCCP subcomplex consists of two dimers of BC held together by four BCCPs, thus having a BCCP:BC ratio of 1:1 (Broussard et al, 2013). In contrast, proteomics (Ishihama et al, 2008), in vivo labeling (Choi-Rhee and Cronan, 2003), and ribosome profiling studies (Li et al, 2014) have observed a BCCP:BC ratio of 2:1.…”

“…Each lane was separated into 0.5-cm segments and subsequently diced into ;1-mm 3 gel Schematic illustrating the proposed mechanism of BADC inhibition. The BC/ BCCP subcomplex design was made based on the crystal structure in E. coli (Broussard et al, 2013), consisting of two dimers of BC and four BCCP proteins. The BADC proteins compete with BCCP for binding to BC.…”