Crystal structure analysis of a fatty acid double-bond hydratase fromLactobacillus acidophilus

Abstract

Bacteria have evolved mechanisms for the hydrogenation of unsaturated fatty acids. Hydroxy fatty acid formation may be the first step in such a process; however, knowledge of the structural and mechanistic aspects of this reaction is scarce. Recently, myosin cross-reactive antigen was shown to be a bacterial FAD-containing hydratase which acts on the 9Z and 12Z double bonds of C16 and C18 non-esterified fatty acids, with the formation of 10-hydroxy and 10,13-dihydroxy fatty acids. These fatty acid hydratases form a large protein family which is conserved across Gram-positive and Gram-negative bacteria with no sequence similarity to any known protein apart from the FAD-binding motif. In order to shed light on the substrate recognition and the mechanism of the hydratase reaction, the crystal structure of the hydratase from Lactobacillus acidophilus (LAH) was determined by single-wavelength anomalous dispersion. Crystal structures of apo LAH and of LAH with bound linoleic acid were refined at resolutions of 2.3 and 1.8 Å, respectively. LAH is a homodimer; each protomer consists of four intricately connected domains. Three of them form the FAD-binding and substrate-binding sites and reveal structural similarity to three domains of several flavin-dependent enzymes, including amine oxidoreductases. The additional fourth domain of LAH is located at the C-terminus and consists of three α-helices. It covers the entrance to the hydrophobic substrate channel leading from the protein surface to the active site. In the presence of linoleic acid, the fourth domain of one protomer undergoes conformational changes and opens the entrance to the substrate-binding channel of the other protomer of the LAH homodimer. The linoleic acid molecule is bound at the entrance to the substrate channel, suggesting movement of the lid domain triggered by substrate recognition.

A novel unsaturated fatty acid hydratase toward C16 to C22 fatty acids from Lactobacillus acidophilus

“…The MCRA protein family is highly conserved among different bacterial species, including gram-positive and gram-negative bacteria, and some of them have FA hydratase activity ( 12,13,(16)(17)(18)(19)28 ).…”

“…However, the hydratase responsible for the conversion of LA to 13-hydroxycis -9-octadecenoic acid has not yet been identifi ed, and the physiological activities of 13-hydroxy-cis -9-octadecenoic acid are unclear. As for the known bacterial hydratases, the length of the carbon chain in the substrate FA is limited to C16 and C18 ( 1,12,13,(16)(17)(18)(19).…”

Structure‐Based Mechanism of Oleate Hydratase from Elizabethkingia meningoseptica

“…[7], [10] Volkov et al have recently determined the first and so far only crystal structure of a fatty acid double bond hydratase, the enzyme from Lactobacillus acidophilus . [14] This structure, however, did not contain the flavin cofactor bound to the protein.…”

Engineering and application of enzymes for lipid modification, an update

“…Next, Volkov and others were able to reattribute MCRAs (myosin-cross-reactive-antigens) from Streptococcus pyogenes, Bifidobacterium breve and Lactobacilli as ∆9 hydratases [125][126][127]. The first crystallization of a ∆9-hydratase from Lactobacillus acidophilus (LAH) in apo form with (pdb:4IA6) and without a ligand (pdb: 4IA5) followed promptly [128]. LAH is a structural homodimer and possesses a long substrate binding channel for fatty acids with an N-terminal lid domain in each protomer.…”