Structure of the enzyme-acyl carrier protein (ACP) substrate gatekeeper complex required for biotin synthesis

Abstract

Although the pimeloyl moiety was long known to be a biotin precursor, the mechanism of assembly of this C7 α,ω-dicarboxylic acid was only recently elucidated. In Escherichia coli, pimelate is made by bypassing the strict specificity of the fatty acid synthetic pathway. BioC methylates the free carboxyl of a malonyl thioester, which replaces the usual acetyl thioester primer. This atypical primer is transformed to pimeloyl-acyl carrier protein (ACP) methyl ester by two cycles of fatty acid synthesis. The question is, what stops this product from undergoing further elongation? Although BioH readily cleaves this product in vitro, the enzyme is nonspecific, which made assignment of its physiological substrate problematical, especially because another enzyme, BioF, could also perform this gatekeeping function. We report the 2.05-Å resolution cocrystal structure of a complex of BioH with pimeloyl-ACP methyl ester and use the structure to demonstrate that BioH is the gatekeeper and its physiological substrate is pimeloyl-ACP methyl ester.cofactor biosynthesis | esterase | protein-protein interaction R ecent work delineated the assembly pathway of the enigmatic pimeloyl moiety of biotin. Labeling studies in Escherichia coli had shown that pimelate, a C7 α,ω-dicarboxylic acid, is made by head-to-tail incorporation of three intact acetate units with one of the carboxyl groups being derived from CO 2 (1, 2). The differing origins of the carboxyl groups indicated that free pimelate was not a synthetic intermediate. The acetate incorporation pattern was consistent with use of the synthetic pathway that produces the usual monocarboxylic fatty acids. However, synthesis of a dicarboxylic acid using the fatty acid synthetic pathway appeared precluded by the strongly hydrophobic active sites of the fatty acid synthetic enzymes (3), which seemed unlikely to tolerate the charged carboxyl group in place of the usual terminal methyl group. The solution to this conundrum was provided by the characterization of two enzymes, BioC and BioH, which do not directly catalyze pimelate synthesis but instead allow fatty acid synthesis to assemble the pimelate moiety (4). Such circumvention of the specificity of normal fatty acid synthesis begins by BioC (an Omethyltransferase) conversion of the ω-carboxyl group of malonylacyl carrier protein (ACP) to a methyl ester using S-adenosyl-Lmethionine (SAM) as a methyl donor (Fig. 1). Conversion to a methyl ester neutralizes the negative charge and provides a methyl carbon that mimics the methyl ends of normal fatty acyl chains. The malonyl-ACP methyl ester can now enter the fatty acid synthetic pathway where it is condensed with malonyl-ACP by a 3-oxoacyl-ACP synthase in a decarboxylating Claisen reaction to give 3-oxoglutaryl-ACP methyl ester. The methyl ester shielding allows the 3-oxo group to be processed to a methylene group by the standard fatty acid reductase-dehydratase-reductase reaction sequence. The resulting glutaryl-ACP methyl ester would then be elongated to the C7 species, and another r...

Using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters

“…Two enzymes from the biotin pathway, BioC (a SAM-dependent methyltransferase) and BioH (an esterase), hijack fatty acid biosynthesis by methylating malonyl-ACP, then extending it through two rounds of the fatty acid synthase cycle 17 . The crystal structure of an inactive BioH S82A mutant:pimeloyl-ACP methyl ester complex revealed the origin of the acyl-chain length specificity 18 .…”

Brucella BioR Regulator Defines a Complex Regulatory Mechanism for Bacterial Biotin Metabolism

“…Recently, we found that the generation of pimeloyl moiety in earlier steps of biotin synthesis is involved in a modified type II fatty acid biosynthetic pathway in E. coli (6)(7)(8). In the BioC-BioH pathway of pimelate synthesis, BioC methylates malonyl coenzyme A (malonyl-CoA; or ACP) and gives a methyl malonyl-thioester destined to fatty acid biosynthesis to act as a primer (6,7,9), whereas the bioH gene product demethylates the pimeloyl-ACP methyl ester to form pimeloyl-ACP after two rounds of the fatty acid elongation cycle (6,7,10). The subsequent four-step pathway functions in assembling the double rings of the biotin molecule (6-8) (Fig.…”

Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle

“…7C), which, in combination with the inherent flexibility of the carrier arm, would allow the carrier arm to be accommodated within active sites of different structure. A paucity of carrier arm interactions with the partner enzyme active site has also been observed for phosphopantetheine-containing carrier proteins, suggesting that this strategy is a general feature of carrier protein interactions (6,(29)(30)(31).…”