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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...

Conjugated linoleic acids (CLAs) affect body fat gain, carcinogenesis, insulin resistance, and lipid peroxidation in mammals. Several isomers of CLA exist, of which the (9Z, 11E) and (10E, 12Z) isomers have beneficial effects on human metabolism but are scarce in foods. Bacterial polyunsaturated fatty acid isomerases are promising biotechnological catalysts for CLA production. We describe six crystal structures of the Propionibacterium acnes polyunsaturated fatty acid isomerase PAI in apo-and product-bound forms. The three-domain flavoprotein has previously undescribed folds outside the FAD-binding site. Conformational changes in a hydrophobic channel toward the active site reveal a unique gating mechanism for substrate specificity. The geometry of the substratebinding site explains the length preferences for C18 fatty acids. A catalytic mechanism for double-bond isomerization is formulated that may be altered to change substrate specificity for syntheses of rare CLAs from easily accessible precursors.conjugated linoleic acid ͉ flavoprotein ͉ polyenoic fatty acid isomerase ͉ structure-based mechanism

BackgroundThe aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. MCRA from B. breve NCIMB 702258 was cloned, sequenced and expressed in heterologous hosts (Lactococcus and Corynebacterium) and the recombinant proteins assessed for enzymatic activity against fatty acid substrates.ResultsMCRA catalysed the conversion of palmitoleic, oleic and linoleic acids to the corresponding 10-hydroxy fatty acids, but shorter chain fatty acids were not used as substrates, while the presence of trans-double bonds and double bonds beyond the position C12 abolished hydratase activity. The hydroxy fatty acids produced were not metabolised further. We also found that heterologous Lactococcus and Corynebacterium expressing MCRA accumulated increasing amounts of 10-HOA and 10-HOE in the culture medium. Furthermore, the heterologous cultures exhibited less sensitivity to heat and solvent stresses compared to corresponding controls.ConclusionsMCRA protein in B. breve can be classified as a FAD-containing double bond hydratase, within the carbon-oxygen lyase family, which may be catalysing the first step in conjugated linoleic acid (CLA) production, and this protein has an additional function in bacterial stress protection.

The catalytic mechanism of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) is explored by kinetic, spectroscopic, and thermodynamic studies. The PAI-catalyzed double bond isomerization takes place by selective removal of the pro-R hydrogen from C-11 followed by suprafacial transfer of this hydrogen to C-9 as shown by conversion of C-9-deuterated substrate isotopologs. Data on the midpoint potential, photoreduction, and cofactor replacement suggest PAI to operate via an ionic mechanism with the formation of FADH 2 and linoleic acid carbocation as intermediates. In line with this proposal, no radical intermediates were detected neither by stopped flow absorption nor by EPR spectroscopy. The substrate preference toward free fatty acids is determined by the interaction between Arg-88 and Phe-193, and the reaction rate is strongly affected by replacement of these amino acids, indicating that the efficiency of the hydrogen transfer relies on a fixed distance between the free carboxyl group and the N-5 atom of FAD. Combining data obtained for PAI from the structural studies and experiments described here suggests that at least two different prototypical active site geometries exist among polyunsaturated fatty acid double bond isomerases. Conjugated linoleic acid (CLA)3 refers to the isomers of linoleic acid (LA, 18:2⌬ 9Z,12Z ; x:y⌬ z denotes a fatty acid with x carbons and y double bonds in position z counting from the carboxyl end) with two conjugated double bonds in various geometrical configurations. It originates from metabolism of anaerobic ruminal bacteria (1-3). Chemically, isomerization of LA to CLA is overall a non-redox process and does not involve oxygen, implying an unique mechanism for all polyunsaturated fatty acid (PUFA) double bond isomerases (4). The first of such isomerases was characterized from Butiryvibrio fibrisolvens by Tove and co-workers (1, 2), who demonstrated that this enzyme prefers C-18 fatty acids and produces (9Z,11E)-CLA. Here the abstraction of a hydrogen atom occurs at position C-11 of LA during catalysis, and one solvent-derived proton is found at position C-13 in the product (2). A mechanistically related eukaryotic PUFA isomerase from marine algae Ptilota filicina has been also described (5, 6), but the identity of the product derived from LA has not yet been reported, because this enzyme prefers C-20 PUFA.Recently, we reported the crystal structure of a FAD-dependent PUFA isomerase from Propionibacterium acnes (PAI) in complex with (10E,12Z)-CLA (7) and proposed a viable reaction mechanism involving the transient oxidation of LA by FAD (Fig. 1A). Thus, PAI belongs to a functionally diverse group of flavoenzymes catalyzing reactions with no net redox change (8). Similar to other PUFA isomerases described so far, PAI prefers free fatty acids as substrates (9), which can be rationalized by the crystal structure (7, 10). The carboxylate group of the fatty acid is hydrogen bonded to Arg-88, which itself interacts via -stacking with Phe-193 (Fig. 1B). This Arg/Phe loc...

The biotransformation of linoleic acid (LA) into conjugated linoleic acid (CLA) by microorganisms is a potentially useful industrial process. In most cases, however, the identities of proteins involved and the details of enzymatic activity regulation are far from clear. Here we summarize available data on the reaction mechanisms of CLA-producing enzymes characterized until now, from Butyrivibrio fibrisolvens, Lactobacillus acidophilus, Ptilota filicina, and Propionibacterium acnes. A general feature of enzymatic LA isomerization is the protein-assisted abstraction of an aliphatic hydrogen atom from position C-11, while the role of flavin as cofactor for the double bond activation in CLA-producing enzymes is also discussed with regard to the recently published three-dimensional structure of an isomerase from P. acnes. Combined data from structural studies, isotopic labeling experiments, and sequence comparison suggest that at least two different prototypical active site geometries occur among polyunsaturated fatty acid (PUFA) double bond isomerases.

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The polyenoic fatty-acid isomerase from Propionibacterium acnes (PAI) catalyzes the double-bond isomerization of linoleic acid to conjugated linoleic acid, which is a dairy-or meat-derived fatty acid in the human diet. PAI was overproduced in Escherichia coli and purified to homogeneity as a yellowcoloured protein. The nature of the bound cofactor was analyzed by absorption and fluorescence spectroscopy. Single crystals of PAI were obtained in two crystal forms. Cubic shaped crystals belong to space group I2 1 3, with a unit-cell parameter of 160.4 Å , and plate-like crystals belong to the monoclinic space group C2, with unit-cell parameters a = 133.7, b = 60.8, c = 72.2 Å , = 115.8 . Both crystal forms contain one molecule per asymmetric unit and diffract to a resolution of better than 2.0 Å . Initial phases were obtained by SIRAS from inhouse data from a cubic crystal that was soaked with an unusually low KI concentration of 0.25 M.