Acetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded β-barrel of the partially resolved BCCP domain. Disruption of β-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTβ-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTβ to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the \"swing domain model\" where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.

Methylcrotonyl-CoA carboxylase (MCCC) is a biotin dependent enzyme, that plays a crucial role in leucine metabolism. The enzyme comprises a biotin carboxylase (BC), a carboxyltransferase (CT), and a biotin carboxyl carrier protein (BCCP) domain. MCCC is synthesized as an apo-protein, and is posttranslationally modified at a lysine residue, conserved in the biotin carboxyl carrier protein (BCCP) domain. In order to understand the structure, function and interactions of L. major MCCC, we have expressed and characterized its domains. Here we report the complete chemical shift assignments of MCCC BCCP domain of L. major. Furthermore, we have used the assignments to generate a model of the same, using CS-Rosetta. We have also followed its chemical shift perturbations upon biotin modification. Changes were observed at the lysine 51 amide, that undergoes biotin modification, and a few others present in its immediate neighborhood.

Terpenoids are the most diverse natural products with many industrial applications and are all synthesized from simple precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, IPP is synthesized by two distinct metabolic pathways - cytosolic mevalonate (MVA) pathway for C15 sesquiterpene and C30 triterpene, and plastidic methylerythritol phosphate (MEP) pathway for C10 monoterpene and C20 diterpene. A number of studies have altered the metabolic gene expressions in either the MVA or MEP pathway to increase terpene production; however, it remains unknown if the alteration of the acetyl-CoA pool in plastid fatty acid biosynthesis can influence terpenoid flux. Here, we focused on the fact that acetyl-CoA is the precursor for both fatty acid biosynthesis in plastid and terpene biosynthesis in cytosol, and the metabolic impact of increased plastidic acetyl-CoA level on the cytosolic terpene biosynthesis was investigated. In tobacco leaf infiltration studies, the acetyl-CoA carboxylase complex (the enzyme supplying malonyl-CoA in plastid) was partially inhibited by overexpressing the inactive form of biotin carboxyl carrier protein (BCCP) by a negative dominant effect. Overexpression of BCCP showed 1.4-2.4-fold increase of sesquiterpenes in cytosol; however, surprisingly overexpression of BCCP linked to truncated HMG-CoA reductase (tHMGR) by a cleavable peptide 2A showed 20-40-fold increases of C15 sesquiterpenes (α-bisabolol, amorphadiene, and valerenadiene) and a 6-fold increase of C30 β-amyrin. α-Bisabolol and β-amyrin production reached 28.8 mg g-1 and 9.8 mg g-1 dry weight, respectively. Detailed analyses showed that a large increase in flux was achieved by the additive effect of BCCP- and tHMGR-overexpression, and an enhanced tHMGR activity by 2A peptide tag. Kinetic analyses showed that tHMGR-2A has a three-fold higher kcat value than tHMGR. The tHMGR-2A-BCCP1 co-expression strategy in this work provides a new insight into metabolic cross-talks and can be a generally applicable approach to over-produce sesqui- and tri-terpene in plants.

Antimicrobial peptides (AMPs) hold great promise as potential therapeutic approach for curing of infectious diseases. Prokaryotic protein expression renders high scalability with an effective purification of several heterogeneous proteins. However, it might be inappropriate for recombinant AMPs expression thereby its antimicrobial activity against the host cells. Several fusion partners demonstrated antimicrobial activity neutralization of AMPs expression and purification in Escherichia coli. In order to improve the antimicrobial effect, several hybrid AMPs have been designed and developed. As expected to increase the antimicrobial activity, a dimeric form of porcine protegrin-1 (PG-1) and human LL-37-linker-histatin-5 (LL-37-linker-Hst-5) hybrid peptide were alternatively constructed in this study. Hydroxylamine hydrochloride and thrombin cleavage sites were designed for releasing of hybrid peptide and PG-1 dimer from biotin carboxyl carrier protein (BCCP) fusion partner. The full-length AMPs gene was connected down-stream of BCCP gene using the overlap extension-PCR, cloned into pET-28a vector and expressed in E. coli BL21(DE3)pLysS. After IPTG induction, approximately 20% of BCCP-AMPs was expressed as intracytoplasmic inclusion bodies with an expected molecular weight of 24.5kDa. The mean of purified and refolded BCCP-AMPs was 1.5mg/L with 76% purity. The presence of expressed protein was subsequently determined by Western blotting analysis. Finally, radial diffusion assay supported that these peptides displayed functional antimicrobial activity against E. coli and Staphylococcus aureus standard strains. Two novel AMPs established in this study would be potentially developed as extensive intervention for treating of infectious diseases.

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates.

Protein partner exchange plays a key role in regulating many biological switches. Although widespread, the mechanisms dictating protein partner identity and, therefore, the outcome of a switch have been determined for a limited number of systems. The Escherichia coli protein BirA undergoes a switch between posttranslational biotin attachment and transcription repression in response to cellular biotin demand. Moreover, the functional switch reflects formation of alternative mutually exclusive protein:protein interactions by BirA. Previous studies provided a set of alanine-substituted BirA variants with altered kinetic and equilibrium parameters of forming these interactions. In this work, DNase I footprinting measurements were employed to investigate the consequences of these altered properties for the outcome of the BirA functional switch. The results support a mechanism in which BirA availability for DNA binding and, therefore, transcription repression is controlled by the rate of the competing protein:protein interaction. However, occupancy of the transcriptional regulatory site on DNA by BirA is exquisitely tuned by the equilibrium constant governing its homodimerization.