Fatty acid biosynthesis generates saturated fatty acids from acetyl-CoA, which is critical for cellular energy storage and membrane synthesis (Blasio and Balzano, 2021). Although this process has been extensively documented in plants and animals, it has also been identified in microalgal species including Chlamydomonas reinhardtii (Giroud et al., 1988), Nannochloropsis oceanica (Vieler et al., 2012), Phaeodactylum tricornutum (Yang et al., 2013), and Ankistrodesmus sp. (Castro et al., 2017). Ankistrodesmus sp. is a green microalga characterized by its high total lipid content and significant protein accumulation, making it a promising candidate for biotechnological applications, particularly for producing biomass rich in lipids and proteins (Cobos Ruiz et al., 2014).

Acetyl-CoA carboxylase (ACC) is a key enzyme in fatty acid biosynthesis that catalyzes the conversion of acetyl-CoA to malonyl-CoA, an essential reaction in the fatty acid elongation cycle (Bellou et al., 2014). There are two major forms of ACC: homomeric (hmACC), found in the cytosol of yeasts, animals, fungi, and in the plastids of grasses; and heteromeric (htACC), which is predominant in prokaryotes, non-grass plants, and the plastids of algae (Huerlimann and Heimann, 2013, Konishi et al., 1996, Sasaki and Nagano, 2004). In algae, ACC can exist in either heteromeric or homomeric forms depending on the origin of the plastid (Huerlimann and Heimann, 2013). Generally, green and red algae contain heteromeric ACC, whereas algae with secondary plastids contain homomeric ACC. Heteromeric ACC consists of four functional subunits: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and two carboxyltransferase subunits (CT-α and CT-β). In contrast, homomeric ACC combines these subunits into a single polypeptide chain within which different functional regions can be easily identified (Cronan and Waldrop, 2002, Wei and Tong, 2015).

The catalytic mechanism of ACC involves two sequential reactions mediated by cooperative interactions among its subunits. In the first step, the BC subunit carboxylates biotin, which is covalently linked to a conserved lysine residue in BCCP (Salie and Thelen, 2016, Tong, 2013). This reaction requires bicarbonate and ATP hydrolysis, with divalent Mg2+ or Mn2+ cations as cofactors. In the second step, the carboxyl group on biotin is transferred to acetyl-CoA by the CT subunits to form malonyl-CoA (Nikolau et al., 2003, Salie and Thelen, 2016). Structural studies have shown that the binding of BCCP to BC induces conformational changes that increase BC’s affinity for ATP and bicarbonate, thereby enhancing carboxylation. Although the initial model of biotin translocation between BC and CT suggested a “swinging arm” motion, recent studies have suggested a more flexible connection between biotin and BCCP, allowing an extension of up to 16 Å (Tong, 2013, Wei and Tong, 2015). However, the distance between the BC and CT active sites ranges from 55 to 85 Å, challenging this model and suggesting a “domain-swinging” mechanism where both biotin and the BCCP subunit move during catalysis (Tong, 2013, Wei and Tong, 2015).

Structural studies of BC (i.e., the separate subunits found in htACC or the functionally equivalent regions of hmACC) revealed the presence of three domains (A, B and C) (Waldrop et al., 1994). Eukaryotic BC has an additional linker that connects domains A and B (AB-linker), explaining its larger size (∼550 residues) compared to prokaryotic BC subunits (∼450 residues) (Cho et al., 2008, Waldrop et al., 1994). Furthermore, an important difference between eukaryotic and prokaryotic BCs lies in the orientational changes in the B-domain with respect to the rest of the structure during catalysis (Chou et al., 2009, Mochalkin et al., 2008). In yeast BC, the B-domain remains in a closed conformation, even without ATP at the active site (Cho et al., 2008, Wei and Tong, 2015). However, bacterial BC undergoes a significant conformational shift in the B-domain upon ATP binding, transitioning from an open to a closed state, similar to other members of the ATP-grasp superfamily (Chou et al., 2009, Thoden et al., 2000). ATP binding regulates BC activity by adopting a conformation that is conducive to biotin carboxylation. In the absence of biotin, ATP binds in a non-productive manner, preventing its reaction with bicarbonate and avoiding futile phosphate hydrolysis (Mochalkin et al., 2008).

Biotin carboxyl carrier protein (BCCP) is a pivotal subunit of acetyl-CoA carboxylase (ACC) that facilitates the transfer of the activated carboxyl group between the biotin carboxylase (BC) and carboxyltransferase (CT) subunits during catalysis (Chapman-Smith and Cronan, 1999, Healy et al., 2010). Structurally, BCCP is defined by a conserved β-sandwich fold that stabilizes the protein and enables covalent biotin binding through a highly conserved lysine residue (Lys122 in E. coli) typically located in a flexible loop region (Chapman-Smith and Cronan, 1999, Healy et al., 2010). This lysine is essential for biotinylation, a post-translational modification that is critical for ACC function. Although the three-dimensional crystal structures of the BC and BCCP subunits have been resolved in some bacterial and eukaryotic species (Chou et al., 2009, Diacovich et al., 2004, Mochalkin et al., 2008, Waldrop et al., 1994), their structural characterization in microalgae remains unexplored (Huerlimann and Heimann, 2013). This gap limits the detailed understanding of the inter-domain interactions and molecular dynamics involved in ACC complex formation in microalgae. The present study reports the progress made towards the structural characterization of biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) (and their heterocomplex) from the oleaginous microalgae Ankistrodesmus sp. Using an experimental and computational approach, we observed an ADP-Mg2+-induced conformational change in the crystallographic structure of BC, substitution of highly conserved lysine by glycine in BCCP, and identification of a novel biotin-binding region in an AlphaFold prediction model.