Kinetic, Structural, and Mutational Analysis of Acyl-CoA Carboxylase From Thermobifida fusca YX

Abstract

Acyl-CoA carboxylases (AcCCase) are biotin-dependent enzymes that are capable of carboxylating more than one short chain acyl-CoA substrate. We have conducted structural and kinetic analyses of such an AcCCase from Thermobifida fusca YX, which exhibits promiscuity in carboxylating acetyl-CoA, propionyl-CoA, and butyryl-CoA. The enzyme consists of two catalytic subunits (TfAcCCA and TfAcCCB) and a non-catalytic subunit, TfAcCCE, and is organized in quaternary structure with a A6B6E6 stoichiometry. Moreover, this holoenzyme structure appears to be primarily assembled from two A3 and a B6E6 subcomplexes. The role of the TfAcCCE subunit is to facilitate the assembly of the holoenzyme complex, and thereby activate catalysis. Based on prior studies of an AcCCase from Streptomyces coelicolor, we explored whether a conserved Asp residue in the TfAcCCB subunit may have a role in determining the substrate selectivity of these types of enzymes. Mutating this D427 residue resulted in alterations in the substrate specificity of the TfAcCCase, increasing proficiency for carboxylating acetyl-CoA, while decreasing carboxylation proficiency with propionyl-CoA and butyryl-CoA. Collectively these results suggest that residue D427 of AcCCB subunits is an important, but not sole determinant of the substrate specificity of AcCCase enzymes.

The Effects of Carbon Source and Growth Temperature on the Fatty Acid Profiles of Thermobifida fusca

“…In contrast, several ACCases from Actinomycetes consist of two subunits: the A subunit that encompasses both the BC and BCCP functional domains, and the B subunit that encompasses the CT domain ( Gago et al, 2011 ; Gago et al, 2018 ). Additionally, some Actinomycete ACCases have a third non-catalytic subunit, E, that is needed for proper assembly of the holoenzyme complex ( Shivaiah et al, 2021 ). Moreover, the A and B subunit quaternary organization of biotin enzymes is also common to propionyl-CoA carboxylase ( Tong, 2013 ) and methylcrotonyl-CoA carboxylase ( Song et al, 1994 ; McKean et al, 2000 ; Wurtele and Nikolau, 2000 ), which complicates the sequence-based identification of ACCase in the T. fusca genome.…”

“…Previous studies have experimentally characterized the T. fusca operon (Tfu_2555, Tfu_2556, Tfu_2557) that encodes the B, E, and A subunits of an acyl-CoA carboxylase (AcCCase) ( Shivaiah et al, 2021 ). This enzyme is promiscuous and can carboxylate acetyl-CoA, propionyl-CoA, and butyryl-CoA.…”

“…These enzymes carboxylate acyl-CoA substrates, including acetyl-CoA, and thereby generate malonyl-CoA ( Nikolau et al, 2003 ), which is considered to be the rate-limiting reaction for fatty acid biosynthesis ( Davis et al, 2000 ; Chaturvedi et al, 2021 ). Prior experimental characterization has identified one of these biotin-containing enzymes as being capable of carboxylating acetyl-CoA, propionyl-CoA, or butyryl-CoA ( Shivaiah et al, 2021 ), but whether T. fusca encodes other proteins that can also carboxylate acetyl-CoA will require similar characterizations.…”

“…The sequenced T. fusca genome was queried with the BLASTP algorithm ( Altschul et al, 1990 ) using query sequences of experimentally confirmed acetyl-CoA carboxylase (ACCase) and Type II fatty acid synthase (FAS) components, either originating from Actinomycetes or from Escherichia coli ( Cronan and Thomas, 2009 ; Shivaiah et al, 2021 ). Additionally, genes were identified using the KEGG genome browser ( Kanehisa et al, 2002 ) based on sequence homology, key processes, and operon organization.…”

Review on the Synthesis and Therapeutic Potential of Pyrido[2,3-d], [3,2-d], [3,4-d] and [4,3-d]pyrimidine Derivatives

“…ACC catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, which is an intermediate substrate. Over the years, ACC inhibitors have attracted great attention in the development of treatments for various human diseases, including microbial infections, metabolic syndrome, obesity, diabetes and cancer [ 22 , 23 ].…”