Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase

Abstract

Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1′-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1′-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO2 from the carboxyphosphate intermediate to biotin.

Hybrid Structure of a Dynamic Single-Chain Carboxylase from Deinococcus radiodurans

“…Carboxybiotin-interacting residues have been identified in the Figure S4. crystal structure of the H. influenzae ACC BC (Broussard et al, 2015) and are structurally conserved in Dra YCC (lysine 235, arginine 290, and arginine 342; Figure 3B), in agreement with a generally conserved BC mechanism.…”

“…The B subdomain is flexibly tethered by two irregular peptide linkers and exhibits increased disorder in the crystal (Figure S4B). Based on homology to the BC domains of E. coli or Haemophilus influenzae ACC, this B subdomain may undergo a hinge-bending motion during substrate binding to act as a lid to the BC active site (Broussard et al, 2015;Thoden et al, 2000). Particularly the highly conserved glycine-rich loop (lysine 157-methionine 167) in the B subdomain might be involved in direct ligand contacts, with lysine 157 presumably interacting with one of the a-phosphoryl oxygens of MgATP ( Figure 3A).…”

Crystal structure of the essential biotin‐dependent carboxylase AccA3 from Mycobacterium tuberculosis

“…3B). The residue in this position interacts directly with the bound nucleotide and is expected to influence the properties of the loop and the residues directly downstream which line the biotin‐binding site, for example, K247, the equivalent residue of K238 in the E. coli protein, shown to influence both the K m for ATP as well as being involved in the carboxylation reaction 12, 47, 48. The substitution to tyrosine is likely to change the properties of both the nucleotide‐binding site and the active site‐bridging loop.…”

“…The AccD transcarboxylase enzyme then transfers the CO 2 from carboxybiotin to the CoA ester substrate 6, 12, 13.…”

Single‐particle analysis of urea amidolyase reveals its molecular mechanism

“…The conversion of urea begins at its C‐terminal urea carboxylase (UC) domain, processing the carboxylation of urea to generate the intermediate product allophanate, which then presumably diffuses into the N‐terminal allophanate hydrolyse (AH) domain to be further hydrolyzed into ammonium . The UC domain belongs to the biotin‐dependent carboxylase family which share common features of domain organization . They all consist of biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT) domains, but in different arrangements (Figure a).…”