The myosin cross-reactive antigen (MCRA) protein family is highly conserved among different bacterial species ranging from Gram-positive to Gram-negative bacteria. Besides their ubiquitous occurrence, knowledge about the biochemical and physiological function of MCRA proteins is scarce. Here, we show that MCRA protein from Streptococcus pyogenes M49 is a FAD enzyme, which acts as hydratase on (9Z)-and (12Z)-double bonds of C-16, C-18 non-esterified fatty acids. Products are 10-hydroxy and 10,13-dihydroxy fatty acids. Kinetic analysis suggests that FAD rather stabilizes the active conformation of the enzyme and is not directly involved in catalysis. Analysis of S. pyogenes M49 grown in the presence of either oleic or linoleic acid showed that 10-hydroxy and 10,13-dihydroxy derivatives were the only products. No further metabolism of these hydroxy fatty acids was detected. Deletion of the hydratase gene caused a 2-fold decrease in minimum inhibitory concentration against oleic acid but increased survival of the mutant strain in whole blood. Adherence and internalization properties to human keratinocytes were reduced in comparison with the wild type. Based on these results, we conclude that the previously identified MCRA protein can be classified as a FAD-containing double bond hydratase, within the carbon-oxygen lyase family, that plays a role in virulence of at least S. pyogenes M49.In 1994, the first member of the MCRA 5 protein family was identified in Streptococcus pyogenes as the result of a screening for antigens recognized by acute rheumatic fever sera. Its amino acid sequence did not exhibit significant similarity to any streptococcal protein with a known function but was conserved among pathogenic groups A, C, and G of Streptococci (1).A BLAST search with the MCRA protein sequence reveals more than 148 conserved sequences across different Grampositive and Gram-negative bacteria (supplemental Fig. S1). MCRA genes are not a part of any bacterial operon. Despite such conservation level only for two members of the family so far, biochemical features have been assigned: MCRA from Lactobacillus reuteri PYR8 was suggested to be a (9Z,11E)-conjugated linoleic acid (CLA)-forming isomerase (2), and only recently hydratase activity was shown for MCRA of Pseudomonas sp. strain 3266 (3).To analyze biochemical properties and physiological functions of MCRA and their more ubiquitous activity as fatty acid hydratase, we have chosen S. pyogenes M49 as a representative of the group A streptococci (GAS). GAS species exclusively colonize humans and cause a wide range of primary infections of the skin, throat, and other mucosal surfaces, including pharyngitis and impetigo (4), and hence have a vast medical importance. We show that S. pyogenes MCRA is a FAD-containing hydratase that adds water to (9Z)-and (12Z)-double bonds of C-16 and C-18 fatty acids. Using a gene deletion strain, we show that the hydroxylated fatty acids are not further metabolized. Importantly, the mutant strain showed alteration in virulence properties, s...

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.

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