Application-Oriented Marine Isomerases in Biocatalysis.

The class EC 5.xx, a group of enzymes that interconvert optical, geometric, or positional isomers are interesting biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. This class, named "isomerases," can transform cheap biomolecules into expensive isomers with suitable stereochemistry useful in synthetic medicinal chemistry, and interesting cases of production of l-ribose, d-psicose, lactulose, and d-phenylalanine are known. However, in two published reports about potential biocatalysts of marine origin, isomerases are hardly mentioned. Therefore, it is of interest to deepen the knowledge of these biocatalysts from the marine environment with this specialized in-depth analysis conducted using a literature search without time limit constraints. In this review, the focus is dedicated mainly to example applications in biocatalysis that are not numerous confirming the general view previously reported. However, from this overall literature analysis, curiosity-driven scientific interest for marine isomerases seems to have been long-standing. However, the major fields in which application examples are framed are placed at the cutting edge of current biotechnological development. Since these enzymes can offer properties of industrial interest, this will act as a promoter for future studies of marine-originating isomerases in applied biocatalysis.

Structural, Kinetic, and Mechanistic Analysis of an Asymmetric 4-Oxalocrotonate Tautomerase Trimer.

A 4-oxalocrotonate tautomerase (4-OT) trimer has been isolated from Burkholderia lata, and a kinetic, mechanistic, and structural analysis has been performed. The enzyme is the third described oligomer state for 4-OT along with a homo- and heterohexamer. The 4-OT trimer is part of a small subset of sequences (133 sequences) within the 4-OT subgroup of the tautomerase superfamily (TSF). The TSF has two distinct features: members are composed of a single ß-α-ß unit (homo- and heterohexamer) or two consecutively joined ß-α-ß units (trimer) and generally have a catalytic amino-terminal proline. The enzyme, designated as fused 4-OT, functions as a 4-OT where the active site groups (Pro-1, Arg-39, Arg-76, Phe-115, Arg-127) mirror those in the canonical 4-OT from Pseudomonas putida mt-2. Inactivation by 2-oxo-3-pentynoate suggests that Pro-1 of fused 4-OT has a low p Ka enabling the prolyl nitrogen to function as a general base. A remarkable feature of the fused 4-OT is the absence of P3 rotational symmetry in the structure (1.5 Å resolution). The asymmetric arrangement of the trimer is not due to the fusion of the two ß-α-ß building blocks because an engineered "unfused" variant that breaks the covalent bond between the two units (to generate a heterohexamer) assumes the same asymmetric oligomerization state. It remains unknown how the different active site configurations contribute to the observed overall activities and whether the asymmetry has a biological purpose or role in the evolution of TSF members.

Optimization of continuous purification of recombinant patchoulol synthase from Escherichia coli with membrane adsorbers.

The natural production of patchouli oil in developing countries cannot meet the increasing demand any more. This leads to socioecological consequences, such as the use of arable land, which is actually intended for food. Hence, the world market price increased up to $150/kg. An alternative is the biotechnological production of patchouli oil using a multiproduct sesquiterpene synthase, the patchoulol synthase (PTS). Here, we report the optimization of recombinant PTS purification from Escherichia coli lysate using continuous immobilized metal affinity chromatography. First, the purification conditions of the batch process were optimized in regard to optimal buffer composition and optimized chromatographic conditions. The best purification result was achieved with Co2+ -immobilized metal affinity chromatography (Sartobind® IDA 75) with a triethanolamine buffer at pH 7, 0.5 M NaCl, 10% [vol/vol] glycerol, 5 mM MgCl2 and 250 mM imidazole for product elution. This optimized method was then transferred to a continuous chromatography system using three membrane adsorber units (surface of 75 cm2 each). Within 1.5 hr in total, 4.55 mg PTS with a final purity of 98% and recovery of 68% could be gained. The purified enzyme was used to produce 126 mg/L (-)-patchoulol from farnesyl pyrophosphate. Here, for the first time bioactive PTS was successfully purified using membrane adsorbers in a continuous downstream process.

Ten-Minute Protein Purification and Surface Tethering for Continuous-Flow Biocatalysis.

Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused Hisn -tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this "reaction-ready" system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.

Non-monotonic course of protein solubility in aqueous polymer-salt solutions can be modeled using the sol-mxDLVO model.

Protein purification is often performed using cost-intensive chromatographic steps. To discover economic alternatives (e.g., crystallization), knowledge on protein solubility as a function of temperature, pH, and additives in solution as well as their concentration is required. State-of-the-art models for predicting protein solubility almost exclusively consider aqueous salt systems, whereas "salting-in" and "salting-out" effects induced by the presence of an additional polymer are not considered. Thus, we developed the sol-mxDLVO model. Using this newly developed model, protein solubility in the presence of one salt and one polymer, especially the non-monotonic course of protein solubility, could be predicted. Systems considered included salts (NaCl, Na-p-Ts, (NH(4))(2) SO(4)) and the polymer polyethylene glycol (MW: 2000 g/mol, 12000 g/mol) and proteins lysozyme from chicken egg white (pH 4 to 5.5) and D-xylose ketol-isomerase (pH 7) at 298.15 K. The results show that by using the sol-mxDLVO model, protein solubility in polymer-salt solutions can be modeled in good agreement with the experimental data for both proteins considered. The sol-mxDLVO model can describe the non-monotonic course of protein solubility as a function of polymer concentration and salt concentration, previously not covered by state-of-the-art models.

OPDA isomerase GST16 is involved in phytohormone detoxification and insect development.

12-Oxophytodienoic acid (OPDA), a well-known phytohormone of the jasmonate family, has a reactive α,ß-unsaturated carbonyl structure which easily adds cellular nucleophiles (Michael addition), making OPDA potentially toxic for herbivores. The glutathione S-transferase GST16 inactivates 12-OPDA in the insect gut by isomerization to inactive iso-OPDA. Quantitative tissue expression analysis showed that HarmGST16 transcripts were present in most larval tissues, including those of the midgut, fatbody and Malpighian tubules. Activity assays confirmed the presence of an active enzyme. Interestingly, feeding different diets to Helicoverpa armigera influenced gst16 expression levels in various tissues, and larvae fed wild-type tobacco leaves had reduced gst16 mRNA levels. The temporal expression of HarmGST16 during larval development was high in the second instar and reduced during the third, fourth and fifth instars. Plant-mediated RNA interference silencing of HarmGST16 retarded larval growth of H. armigera. Injecting cis-OPDA into the hemolymph of larvae caused premature pupation. This result, as well as the finding that GST16 influenced the growth of insects, suggests that GST16 may play an important role in larval development.

Expression, purification and activity assay of a patchoulol synthase cDNA variant fused to thioredoxin in Escherichia coli.

Probing a cDNA library extracted from Pogostemon cablin (Indian Patchouli) with gene specific primers, a variant of patchoulol synthase PTS (GenBank acc. No.: AY508730) was amplified, cloned, and sequenced. The amino acid sequence deduced from the cloned cDNA exhibited a sequence variation of 3.4% compared to the annotated sequence. The enzyme variant tended to form inclusion bodies when expressed in Escherichia coli. The coding sequence was fused to the T7-tag, His-tag and to thioredoxin. Constructs were expressed in three different E. coli expression strains, with several strain/construct combinations yielding soluble enzyme. By fusion to thioredoxin and careful codon optimization of the eukaryotic sequence, soluble expression could be improved on average by 42% in comparison to an unoptimized, His-tagged construct. The thioredoxin-fused protein was successfully purified using a one-step Co(2+)-IMAC purification procedure. Bioactivity assays using prepared farnesyl diphosphate (FDP) in milliliter-scale batch reactions, showed activity of the fused enzyme even with thioredoxin attached. The product spectrum of the enzyme was compared to patchouli oil standards by GC-MS and the main products were identified. Interestingly, the variant showed a shift in product spectrum with germacrene A being the most abundant product instead of patchouli alcohol. In silico structural modeling shows a possible chemical and structural change in the active site of the enzyme, which might be responsible for the shift in product composition.

Comparison of enzymatic activity of two linoleic acid isomerases expressed in E. coli.

Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of octadecadienoic fatty acid with conjugated double bonds. CLA possesses many important physiological functions and it can be produced from linoleic acid (LA) by LA isomerases. In this report, we first cloned the genes encoding LA isomerases: C12 isomerases and C9 isomerase, then transformed the recombinant plasmids into Escherichia coli TOP10 and induced E. coli with IPTG (isopropylthio-ß-D-galactoside) to express the recombinant proteins. Next, we purified the isomerases using a HisTrap™ HP column, followed with the analysis by SDS-PAGE or Western blot. Finally, we compared their enzymatic activity by biotransformation of LA into CLA. Plasmids containing LA isomerase genes were successfully constructed. LA isomerases were found expressed in E. coli, and the molecular weight was 64 KD for C12 LA isomerase and 55 KD for C9 LA isomerase. The enzyme activity (9.93 ± 0.01 U/ml for C12 LA isomerase and 8.12 ± 0.02 U/ml for C9 LA isomerase) of both LA isomerases reached the highest when IPTG concentration is 0.2 mM and the induction time is 18 h. After purification, C9 LA isomerase was enriched in peak 4 and C12 LA isomerase was enriched in peak 3. Optimum pH for C9 LA and C12 LA isomerases were 7.5 and 7.0 separately, and optimum temperatures was 37 °C for highest concentration of CLA. The work may provide theoretical significance for an effective production process of CLA for the medical and nutritional purposes.

FabQ, a dual-function dehydratase/isomerase, circumvents the last step of the classical fatty acid synthesis cycle.

In the classical anaerobic pathway of unsaturated fatty acid biosynthesis, that of Escherichia coli, the double bond is introduced into the growing acyl chain by the FabA dehydratase/isomerase. Another dehydratase, FabZ, functions in the chain elongation cycle. In contrast, Aerococcus viridans has only a single FabA/FabZ homolog we designate FabQ. FabQ can not only replace the function of E. coli FabZ in vivo, but it also catalyzes the isomerization required for unsaturated fatty acid biosynthesis. Most strikingly, FabQ in combination with E. coli FabB imparts the surprising ability to bypass reduction of the trans-2-acyl-ACP intermediates of classical fatty acid synthesis. FabQ allows elongation by progressive isomerization reactions to form the polyunsaturated fatty acid, 3-hydroxy-cis-5, 7-hexadecadienoic acid, both in vitro and in vivo. FabQ therefore provides a potential pathway for bacterial synthesis of polyunsaturated fatty acids.

Detection of platypus-type L/D-peptide isomerase activity in aqueous extracts of papaya fruit.

Peptide isomerase catalyses the post-translational isomerisation of the L: - to the D: -form of an amino acid residue around the N/C-termini of substrate peptides. To date, some peptide isomerases have been found in a limited number of animal secretions and cells. We show here that papaya extracts have weak peptide isomerase activity. The activity was detected in each 30-100 kDa fraction of the flesh and the seed extracts of unripe and ripe papaya fruit. The definitive activity was confirmed in the ripe papaya extracts, but even then it was much less active than that of the other peptide isomerases previously reported. The activity was markedly inhibited by methanol, and partly so by amastatin and diethyl pyrocarbonate. This is the first report of peptide isomerase activity in a plant and suggests that perhaps every living organism may have some peptide isomerase activity.

Styrene oxide isomerase of Rhodococcus opacus 1CP, a highly stable and considerably active enzyme.

Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium Rhodococcus opacus 1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg(-1) represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 µmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process.

Expression and purification of the p75 neurotrophin receptor transmembrane domain using a ketosteroid isomerase tag.

BACKGROUND: Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr). RESULT: The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy. CONCLUSIONS: These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.

Linoleic acid isomerase in Lactobacillus plantarum AKU1009a proved to be a multi-component enzyme system requiring oxidoreduction cofactors.

Linoleic acid isomerase in Lactobacillus plantarum was found to be a novel multi-component enzyme system widespread in membrane and soluble fractions. The isomerization reaction involved a hydration step, 10-hydroxy-12-octadecenoic acid production from linoleic acid, as part of the reaction, and the hydration reaction was catalyzed by the membrane fraction. Both membrane and soluble fractions were required for the whole isomerization reaction, i.e., conjugated linoleic acid (CLA) production from linoleic acid, and for CLA production from 10-hydroxy-12-octadecenoic acid, a reaction intermediate. The multi-component enzyme system was inhibited by o-phenanthroline, and divalent metal ions such as Ni(2+) and Co(2+) restored activity. Metal oxides such as VO(4)(3+), MoO(4)(2+), and MnO(4)(2+) enhanced activity. The multi-component enzyme systems required oxidoreduction cofactors such as NADH together with FAD or NADPH for total activity.

Intermediacy of eudesmane cation during catalysis by aristolochene synthase.

Aristolochene synthase from Penicillium roqueforti (PR-AS) catalyzes the formation of the bicyclic sesquiterpene (+)-aristolochene (5) from farnesyl diphosphate (1, FDP) in two mechanistically distinct cyclization reactions. The first reaction transforms farnesyl diphosphate to the uncharged intermediate (S)-(-)-germacrene A (3) through a macrocyclization process that links C1 and C10 upon magnesium ion-assisted diphosphate ester activation. In the second reaction mediated by PR-AS, a protonation induced cyclization has been suggested to generate the highly reactive trans-fused eudesmane cation 4 as a consequence of the precise folding of the enzyme-bound germacrene A intermediate. This contribution describes the use of the transition state analogue inhibitor 4-aza-eudesm-11-ene to explore the intermediacy of cation 4 as an on-path intermediate in the biosynthesis of aristolochene. 4-Aza-eudesm-11-ene as the hydrochloride salt 6 was stereospecifically synthesized in seven steps and 37% overall yield starting from chiral enamine 9. The synthetic sequence featured a highly regio- and stereoselective deracemization reaction of 9 that gave rise to the corresponding Michael adduct in >95% diastereomeric excess as evidenced by optical rotation and NMR measurements. 6 acts as a potent competitive inhibitor of PR-AS (K(i) = 0.35 +/- 0.12 microM) independent of the presence of diphosphate (K(i) = 0.24 +/- 0.09 microM). The failure of exogenous PP(i) to enhance the binding affinity of 6 for PR-AS could be interpreted against an eudesmyl cation/diphosphate anion pair mechanism as the enzymatic strategy to stabilize the highly reactive eudesmane cation 4. In addition, these observations seem to rule out simple favorable electrostatic and/or hydrogen bonding interactions between the active site anchored diphosphate ion and the ammonium ion 6 as the binding mode. Ammonium ion 6 seems to act as a genuine mimic of eudesmane cation (4) that most likely binds the active site of PR-AS in a productive conformation resembling that adapted by 4 during the PR-AS-catalyzed synthesis of 5.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the small subunit of isopropylmalate isomerase (Rv2987c) from Mycobacterium tuberculosis.

Two C-terminally truncated variants of the small subunit of Mycobacterium tuberculosis isopropylmalate isomerase (Rv2987c; LeuD), LeuD_1-156 and LeuD_1-168, have been cloned, heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized. The crystals of LeuD_1-156 belonged to the hexagonal system (space group P6(1)22 or P6(5)22) with up to four subunits in the asymmetric unit, whereas the crystals of LeuD_1-168 belonged to the monoclinic system (space group P2(1)) with two subunits in the asymmetric unit. Both crystals diffracted X-rays to beyond 2.0 A resolution and were suitable for further crystallographic analysis.

Study of anoxic and oxic cholesterol metabolism by Sterolibacterium denitrificans.

The initial enzymes and genes involved in the anoxic metabolism of cholesterol were studied in the denitrifying bacterium Sterolibacterium denitrificans Chol-1S(T). The second enzyme of the proposed pathway, cholest-4-en-3-one-Delta1-dehydrogenase (AcmB), was partially purified. Based on amino acid sequence analysis, a gene probe was derived to screen a cosmid library of chromosomal DNA for the acmB gene. A positive clone comprising a 43-kbp DNA insert was sequenced. In addition to the acmB gene, the DNA fragment harbored the acmA gene, which encodes the first enzyme of the pathway, cholesterol dehydrogenase/isomerase. The acmA gene was overexpressed, and the recombinant dehydrogenase/isomerase was purified. This enzyme catalyzes the predicted transformation of cholesterol to cholest-4-en-3-one. S. denitrificans cells grown aerobically with cholesterol exhibited the same pattern of soluble proteins and cell extracts formed the same 14C-labeled products from [14C]cholesterol as cells that were grown under anoxic, denitrifying conditions. This is especially remarkable for the late products that are formed by anaerobic hydroxylation of the cholesterol side chain with water as the oxygen donor. Hence, this facultative anaerobic bacterium may use the anoxic pathway lacking any oxygenase-dependent reaction also under oxic conditions. This confers metabolic flexibility to such facultative anaerobic bacteria.

Characterization of a novel D-lyxose isomerase from Cohnella laevoribosii RI-39 sp. nov.

A newly isolated bacterium, Cohnella laevoribosii RI-39, could grow in a defined medium with L-ribose as the sole carbon source. A 21-kDa protein isomerizing L-ribose to L-ribulose, as well as D-lyxose to D-xylulose, was purified to homogeneity from this bacterium. Based on the N-terminal and internal amino acid sequences of the purified enzyme obtained by N-terminal sequencing and quantitative time of flight mass spectrometry-mass spectrometry analyses, a 549-bp gene (lyxA) encoding D-lyxose (L-ribose) isomerase was cloned and expressed in Escherichia coli. The purified endogenous enzyme and the recombinant enzyme formed homodimers that were activated by Mn(2+). C. laevoribosii D-lyxose (L-ribose) isomerase (CLLI) exhibits maximal activity at pH 6.5 and 70 degrees C in the presence of Mn(2+) for D-lyxose and L-ribose, and its isoelectric point (pI) is 4.2 (calculated pI, 4.9). The enzyme is specific for D-lyxose, L-ribose, and D-mannose, with apparent K(m) values of 22.4 +/- 1.5 mM, 121.7 +/- 10.8 mM, and 34.0 +/- 1.1 mM, respectively. The catalytic efficiencies (k(cat)/K(m)) of CLLI were 84.9 +/- 5.8 mM(-1) s(-1) for D-lyxose (V(max), 5,434.8 U mg(-1)), 0.2 mM(-1) s(-1) for L-ribose (V(max), 75.5 +/- 6.0 U mg(-1)), and 1.4 +/- 0.1 mM(-1) s(-1) for D-mannose (V(max), 131.8 +/- 7.4 U mg(-1)). The ability of lyxA to permit E. coli cells to grow on D-lyxose and L-ribose and homology searches of other sugar-related enzymes, as well as previously described sugar isomerases, suggest that CLLI is a novel type of rare sugar isomerase.

Metabolism of 4-amino-3-hydroxybenzoic acid by Bordetella sp. strain 10d: A different modified meta-cleavage pathway for 2-aminophenols.

Bordetella sp. strain 10d metabolizes 4-amino-3-hydroxybenzoic acid via 2-hydroxymuconic 6-semialdehyde. Cell extracts from 4-amino-3-hydroxybenzoate-grown cells showed high NAD(+)-dependent 2-hydroxymuconic 6-semialdehyde dehydrogenase, 4-oxalocrotonate tautomerase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase activities, but no 2-hydroxymuconic 6-semialdehyde hydrolase activity. These enzymes involved in 4-amino-3-hydroxybenzoate metabolism were purified and characterized. When 2-hydroxymuconic 6-semialdehyde was used as substrate in a reaction mixture containing NAD(+) and cell extracts from 4-amino-3-hydroxybenzoate-grown cells, 4-oxalocrotonic acid, 2-oxopent-4-enoic acid, and 4-hydroxy-2-oxovaleric acid were identified as intermediates, and pyruvic acid was identified as the final product. A complete pathway for the metabolism of 4-amino-3-hydroxybenzoic acid in strain 10d is proposed. Strain 10d metabolized 2-hydroxymuconic 6-semialdehyde derived from 4-amino-3-hydroxybenzoic acid via a dehydrogenative route, not via a hydrolytic route. This proposed metabolic pathway differs considerably from the modified meta-cleavage pathway of 2-aminophenol and those previously reported for methyl- and chloro-derivatives.

Isoleucine biosynthesis in Leptospira interrogans serotype lai strain 56601 proceeds via a threonine-independent pathway.

Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded alpha-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower K(m) (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed beta-methyl-d-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and beta-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes.

A new mechanism for anaerobic unsaturated fatty acid formation in Streptococcus pneumoniae.

The anaerobic pathway for unsaturated fatty acid synthesis was established in the 1960s in Escherichia coli. The double bond is introduced into the growing acyl chain by FabA, an enzyme capable of both the dehydration of beta-hydroxydecanoyl-acyl carrier protein (ACP) to trans-2-decenoyl-ACP, and the isomerization of trans-2 to cis-3-decenoyl-ACP. However, there are a number of anaerobic bacteria whose genomes do not contain a fabA homolog, although these organisms nonetheless produce unsaturated fatty acids. We cloned and biochemically characterized a new enzyme in type II fatty acid synthesis from Streptococcus pneumoniae that carries out the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP, but is not capable of catalyzing the dehydration of beta-hydroxy intermediates. This tetrameric enzyme, designated FabM, has no similarity to FabA, but rather is a member of the hydratase/isomerase superfamily. Thus, the branch point in the biosynthesis of unsaturated fatty acids in S. pneumoniae occurs following the formation of trans-2-decenoyl-ACP, in contrast to E. coli where the branch point takes place after the formation of beta-hydroxydecanoyl-ACP.