Myosin Cross-reactive Antigen of Streptococcus pyogenes M49 Encodes a Fatty Acid Double Bond Hydratase That Plays a Role in Oleic Acid Detoxification and Bacterial Virulence

Abstract

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...

Comparison of Biochemical Properties of the Original and Newly Identified Oleate Hydratases from Stenotrophomonas maltophilia

“…The same results were reported in OhyAs from S. pyogenes (10) and M. caseolyticus (12). Thus, apo-OhyAs were shown to be cofactor-dependent enzymes.…”

“…Based on the kinetic analysis of OhyA from S. pyogenes, FAD was involved in the stabilization of the enzyme but not directly involved in catalysis (10). The reaction mechanism of OhyA from E. meningoseptica proposed that FAD was involved in the correct localization of the substrate and amino acids in the active site of OhyA (7).…”

“…The biochemical properties of OhyAs from Elizabethkingia meningoseptica (6,7), Bifidobacterium breve (8,9), Streptococcus pyogenes (10), Lysinibacillus fusiformis (11), Macrococcus caseolyticus (12), Stenotrophomonas maltophilia (13), and Stenotrophomonas nitritireducens (14) have been characterized to date. All OhyAs contain flavin adenine dinucleotide (FAD)-binding motifs.…”

“…Unsaturated fatty acids disrupt the membrane structure in cells because of their distorted structures (17) and inhibit fatty acid-synthesizing enzymes (18). For surviving in unsaturated fatty acid-rich environments, as a detoxification mechanism, bacteria harboring MCRA proteins hydrate unsaturated fatty acids and convert them to hydroxy fatty acids, which are degraded in cells (10).…”

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

“…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).…”