Leishmania major biotin protein ligase forms a unique cross-handshake dimer

Abstract

Biotin protein ligase catalyses the post-translational modification of biotin carboxyl carrier protein (BCCP) domains, a modification that is crucial for the function of several carboxylases. It is a two-step process that results in the covalent attachment of biotin to the ɛ-amino group of a conserved lysine of the BCCP domain of a carboxylase in an ATP-dependent manner. In Leishmania, three mitochondrial enzymes, acetyl-CoA carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, depend on biotinylation for activity. In view of the indispensable role of the biotinylating enzyme in the activation of these carboxylases, crystal structures of L. major biotin protein ligase complexed with biotin and with biotinyl-5′-AMP have been solved. L. major biotin protein ligase crystallizes as a unique dimer formed by cross-handshake interactions of the hinge region of the two monomers formed by partial unfolding of the C-terminal domain. Interestingly, the substrate (BCCP domain)-binding site of each monomer is occupied by its own C-terminal domain in the dimer structure. This was observed in all of the crystals that were obtained, suggesting a closed/inactive conformation of the enzyme. Size-exclusion chromatography studies carried out using high protein concentrations (0.5 mM) suggest the formation of a concentration-dependent dimer that exists in equilibrium with the monomer.

CryoEM reveals oligomeric isomers of a multienzyme complex and assembly mechanics

“…One such multienzyme is propionyl-CoA carboxylase (PCC), which catalyzes the carboxylation of propionyl-CoA to form methylmalonyl-CoA, a precursor to the citric acid cycle intermediate succinyl-CoA ( Halenz and Lane, 1960 ). PCC is found in bacteria ( Huang et al, 2010 ), archaea ( Hou et al, 2015 ), protozoa ( Rajak et al, 2021 ), plants ( Wurtele and Nikolau, 1990 ), and animals ( Huang et al, 2010 ). In humans, inherited mutations in the genes encoding PCC may interfere with multienzyme assembly, leading to catalytic dysfunction ( Jiang et al, 2005 ) and the metabolic disorder known as propionic acidemia ( Grünert et al, 2013 ).…”

CryoEM Reveals Oligomeric Isomers of a Multienzyme Complex and Assembly Mechanics

“…One such multienzyme is propionyl-CoA carboxylase (PCC), which catalyzes the carboxylation of propionyl-CoA to form methylmalonyl-CoA, a precursor to the citric acid cycle intermediate succinyl-CoA [4]. As citric acid cycle is essential to cellular metabolism, PCC is found in bacteria [5], archaea [6], protozoa [7], plants [8], and animals [5]. In humans, inherited mutations in the genes encoding PCC may interfere with multienzyme assembly leading to catalytic dysfunction [9] and the metabolic disorder known as propionic acidemia [10].…”