Only one of the two annotated Lactococcus lactis fabG genes encodes a functional beta-ketoacyl-acyl carrier protein reductase

The small genome of the Gram-positive bacterium Lactococcus lactis ssp. lactis IL1403 contains two genes that encode proteins annotated as homologues of Escherichia coli beta-hydroxyacyl-acyl carrier protein (ACP) reductase. E. coli fabG encodes beta-ketoacyl-acyl carrier protein (ACP) reductase, the enzyme responsible for the first reductive step of the fatty acid synthetic cycle. Both of the L. lactis genes are adjacent to (and predicted to be cotranscribed with) other genes that encode proteins having homology to known fatty acid synthetic enzymes. Such relationships have often been used to strengthen annotations based on sequence alignments. Annotation in the case of beta-ketoacyl-ACP reductase is particularly problematic because the protein is a member of a vast protein family, the short-chain alcohol dehydrogenase/reductase (SDR) family. The recent isolation of an E. coli fabG mutant strain encoding a conditionally active beta-ketoacyl-ACP reductase allowed physiological and biochemical testing of the putative L. lactishomologues. We report that expression of only one of the two L. lactis proteins (that annotated as FabG1) allows growth of the E. coli fabG strain under nonpermissive conditions and restores in vitro fatty acid synthetic ability to extracts of the mutant strain. Therefore, like E. coli, L. lactis has a single beta-ketoacyl-ACP reductase active with substrates of all fatty acid chain lengths. The second protein (annotated as FabG2), although inactive in fatty acid synthesis both in vivo and in vitro, was highly active in reduction of the model substrate, beta-ketobutyryl-CoA. As expected from work on the E. coli enzyme, the FabG1 beta-ketobutyryl-CoA reductase activity was inhibited by ACP (which blocks access to the active site) whereas the activity of FabG2 was unaffected by the presence of ACP. These results seem to be an example of a gene duplication event followed by divergence of one copy of the gene to encode a protein having a new function.     

大肠杆菌3-酮基脂酰ACP还原酶110位天冬酰胺突变后的结构与功能

3-酮基脂酰ACP还原酶催化3-酮基脂酰ACP还原为3-羟基脂酰ACP,是细菌脂肪酸合成反应的关键酶之一.为了明确该酶中110位的保守天冬酰胺残基在酶催化活性和酶结构中的作用,本研究采用基因定点突变和蛋白质表达纯化技术,获得了大肠杆菌3-酮基脂酰ACP还原酶FabG的两个突变蛋白：FabG N110Q和FabG N110L.圆二色谱结果显示,天冬酰胺残基的突变改变了FabG的空间结构,使突变蛋白的α螺旋结构明显增加.以3-酮脂酰ACP为底物的酶活性测定表明,突变蛋白的酶活性均有下降,但残存的酶活性达到了FabG的75%以上.突变蛋白FabG N110Q和FabG N110L具有3-酮基脂酰ACP还原酶的活性,能在体外重建细菌脂肪酸合成反应.对fabG温度敏感突变株的遗传互补分析表明,FabG蛋白110位天冬酰胺突变为谷氨酰胺或亮氨酸后,在一定的条件下仍能互补大肠杆菌的生长.本研究结果提示,FabG 110位的天冬酰胺残基不是参与3-酮基脂酰ACP还原酶催化反应的必需氨基酸,它只是作为结构氨基酸,在维持FabG的空间结构的稳定性方面起作用.     

The Pseudomonas aeruginosa rhlG Gene Encodes an NADPH-Dependent β-Ketoacyl Reductase Which Is Specifically Involved in Rhamnolipid Synthesis

A Pseudomonas aeruginosa gene homologous to the fabG gene, which encodes the NADPH-dependent β-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this fabG homolog (herein called rhlG) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-β-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by the rhlG mutation. We conclude that the P. aeruginosa rhlG gene encodes an NADPH-dependent β-ketoacyl reductase absolutely required for the synthesis of the β-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of rhlG mRNA under different culture conditions is consistent with this conclusion.     

Cofactor-induced conformational rearrangements establish a catalytically competent active site and a proton relay conduit in FabG

beta-Ketoacyl-acyl carrier protein reductase (FabG) is a key component in the type II fatty acid synthase system. The structures of Escherichia coli FabG and the FabG[Y151F] mutant in binary complexes with NADP(H) reveal that mechanistically important conformational changes accompany cofactor binding. The active site Ser-Tyr-Lys triad is repositioned into a catalytically competent constellation, and a hydrogen bonded network consisting of ribose hydroxyls, the Ser-Tyr-Lys triad, and four water molecules creates a proton wire to replenish the tyrosine proton donated during catalysis. Also, a disordered loop in FabG forms a substructure in the complex that shapes the entrance to the active site. A key observation is that the nicotinamide portion of the cofactor is disordered in the FabG[Y151F].NADP(H) complex, and Tyr151 appears to be necessary for high-affinity cofactor binding. Biochemical data confirm that FabG[Y151F] is defective in NADPH binding. Finally, structural changes consistent with the observed negative cooperativity of FabG are described.     

Structure of beta-ketoacyl-[acyl carrier protein] reductase from Escherichia coli: negative cooperativity and its structural basis.

The structure of beta-ketoacyl-[acyl carrier protein] reductase (FabG) from Escherichia coli was determined via the multiwavelength anomalous diffraction technique using a selenomethionine-labeled crystal containing 88 selenium sites in the asymmetric unit. The comparison of the E. coli FabG structure with the homologous Brassica napus FabG.NADP(+) binary complex reveals that cofactor binding causes a substantial conformational change in the protein. This conformational change puts all three active-site residues (Ser 138, Tyr 151, and Lys 155) into their active configurations and provides a structural mechanism for allosteric communication between the active sites in the homotetramer. FabG exhibits negative cooperative binding of NADPH, and this effect is enhanced by the presence of acyl carrier protein (ACP). NADPH binding also increases the affinity and decreases the maximum binding of ACP to FabG. Thus, unlike other members of the short-chain dehydrogenase/reductase superfamily, FabG undergoes a substantial conformational change upon cofactor binding that organizes the active-site triad and alters the affinity of the other substrate-binding sites in the tetrameric enzyme.     

细菌3-酮脂酰ACP还原酶研究进展

3-酮脂酰ACP还原酶(FabG)在细菌中广泛存在并且十分保守,已经发现的所有FabG及其同系物都具有类似的催化活性中心序列,隶属于短链醇脱氢酶/还原酶(SDRs)超家族成员。它是Ⅱ型脂肪酸合成反应中的关键酶,将3-酮脂酰ACP还原为3-羟脂酰ACP多以NADPH作为辅酶。从搜集的文献来看,国内外针对不同细菌中3-酮脂酰ACP还原酶同系物的研究报道体现了其多样性的特点。但是,近年来,该方面的专题综述十分少见。本文主要对3-酮脂酰ACP还原酶的结构特征、在脂肪酸合成和其他方面的生物学功能,以及以该酶为作用靶点的抑菌剂等方面进行概述,以期为将来3-酮脂酰ACP还原酶的深入研究提供理论参考。     

Deciphering the key residues in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein

The type II fatty acid synthase (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials, due to its intrinsic differences from the typeI pathway operating in humans. beta-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction of beta-ketoacyl-ACP to beta-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. In this article, we report intensive studies on the direct interactions of Plasmodium FabG and Plasmodium ACP in solution, in the presence and absence of its cofactor, NADPH, by monitoring the change in intrinsic fluorescence of P.falciparum FabG (PfFabG) and by surface plasmon resonance. To address the issue of the importance of the residues involved in strong, specific and stoichiometric binding of PfFabG to P.falciparum ACP (PfACP), we mutated Arg187, Arg190 and Arg230 of PfFabG. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The affinities of all the PfFabG mutants for acetoacetyl-ACP (the physiological substrate) were reduced to different extents as compared to wild-type PfFabG, but were equally active in biochemical assays with the substrate analog acetoacetyl-CoA. Kinetic analysis and studies of direct binding between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical residues for the PfFabG-PfACP interactions. Our studies thus reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of PfFabG for interactions with PfACP.     

Isolation and characterization of beta-ketoacyl-acyl carrier protein reductase (fabG) mutants of Escherichia coli and Salmonella enterica serovar Typhimurium

FabG, beta-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of beta-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The beta-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.     

Structural Characterisation of FabG from Yersinia pestis,a Key Component of Bacterial Fatty Acid Synthesis

Ketoacyl-acyl carrier protein reductases (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI) has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG), the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.     

The X-ray structure of Brassica napus beta-keto acyl carrier protein reductase and its implications for substrate binding and catalysis

BACKGROUND: beta-Keto acyl carrier protein reductase (BKR) catalyzes the pyridine-nucleotide-dependent reduction of a 3-oxoacyl form of acyl carrier protein (ACP), the first reductive step in de novo fatty acid biosynthesis and a reaction often performed in polyketide biosynthesis. The Brassica napus BKR enzyme is NADPH-dependent and forms part of a dissociable type II fatty acid synthetase (FAS). Significant sequence similarity is observed with enoyl acyl carrier protein reductase (ENR), the other reductase of FAS, and the short-chain alcohol dehydrogenase (SDR) family. RESULTS: The first crystal structure of BKR has been determined at 2.3 A resolution in a binary complex with an NADP(+) cofactor. The structure reveals a homotetramer in which each subunit has a classical dinucleotide-binding fold. A triad of Ser154, Tyr167 and Lys171 residues is found at the active site, characteristic of the SDR family. Overall BKR has a very similar structure to ENR with good superimposition of catalytically important groups. Modelling of the substrate into the active site of BKR indicates the need for conformational changes in the enzyme. CONCLUSIONS: A catalytic mechanism can be proposed involving the conserved triad. Helix alpha6 must shift its position to permit substrate binding to BKR and might act as a flexible lid on the active site. The similarities in fold, mechanism and substrate binding between BKR, which catalyzes a carbon-oxygen double-bond reduction, and ENR, the carbon-carbon double-bond oxidoreductase in FAS, suggest a close evolutionary link during the development of the fatty acid biosynthetic pathway.     

Novel inhibitors of beta-ketoacyl-ACP reductase from Escherichia coli

Bacterial beta-ketoacyl-[acyl carrier protein] (beta-ketoacyl-ACP) reductase (FabG) is a highly conserved and ubiquitously expressed enzyme of the fatty-acid biosynthetic pathway of prokaryotic organisms that catalyzes NADPH-dependent reduction of beta-ketoacyl-ACP intermediates. Therefore, FabG represents an appealing target for the development of new antimicrobial agents. A number of trans-cinnamic acid derivatives were designed and screened for inhibitory activities against FabG from Escherichia coli. These inhibited FabG enzymatic activity with IC(50) values in the microM range, and were used as templates for the subsequent diversification of the chemotype. Introduction of an electron-withdrawing 4-cyano group to the phenol substituent showed improved inhibition over the non-substituted compound. The benzo-[1,3]-dioxol moiety also appeared to be essential for inhibitory activity of trans-cinnamic acid derivatives against FabG from E. coli. To explain the possible binding position, the best inhibitor from the present study was docked in the active site of FabG. The results for the best scoring conformers chosen by the docking programme revealed that cinnamic acid derivatives can be accommodated in the substrate-binding region of the active site, above the nicotinamide moiety of the NADPH cofactor. Additionally, a phage-displayed library of random linear 15-mer peptides was screened against FabG, to identify ligands with the common PPLTXY motif.     

Structure and reactivity of human mitochondrial 2,4-dienoyl-CoA reductase: enzyme-ligand interactions in a distinctive short-chain reductase active site

Fatty acid catabolism by beta-oxidation mainly occurs in mitochondria and to a lesser degree in peroxisomes. Poly-unsaturated fatty acids are problematic for beta-oxidation, because the enzymes directly involved are unable to process all the different double bond conformations and combinations that occur naturally. In mammals, three accessory proteins circumvent this problem by catalyzing specific isomerization and reduction reactions. Central to this process is the NADPH-dependent 2,4-dienoyl-CoA reductase. We present high resolution crystal structures of human mitochondrial 2,4-dienoyl-CoA reductase in binary complex with cofactor, and the ternary complex with NADP(+) and substrate trans-2,trans-4-dienoyl-CoA at 2.1 and 1.75 A resolution, respectively. The enzyme, a homotetramer, is a short-chain dehydrogenase/reductase with a distinctive catalytic center. Close structural similarity between the binary and ternary complexes suggests an absence of large conformational changes during binding and processing of substrate. The site of catalysis is relatively open and placed beside a flexible loop thereby allowing the enzyme to accommodate and process a wide range of fatty acids. Seven single mutants were constructed, by site-directed mutagenesis, to investigate the function of selected residues in the active site thought likely to either contribute to the architecture of the active site or to catalysis. The mutant proteins were overexpressed, purified to homogeneity, and then characterized. The structural and kinetic data are consistent and support a mechanism that derives one reducing equivalent from the cofactor, and one from solvent. Key to the acquisition of a solvent-derived proton is the orientation of substrate and stabilization of a dienolate intermediate by Tyr-199, Asn-148, and the oxidized nicotinamide.     

Key residues responsible for acyl carrier protein and beta-ketoacyl-acyl carrier protein reductase (FabG) interaction

Fatty acid synthesis in bacteria is catalyzed by a set of individual enzymes collectively known as type II fatty-acid synthase. Each enzyme interacts with acyl carrier protein (ACP), which shuttles the pathway intermediates between the proteins. The type II enzymes do not possess primary sequence similarity that defines a common ACP-binding site, but rather are hypothesized to possess an electropositive/hydrophobic surface feature that interacts with the electronegative/hydrophobic residues along helix alpha2 of ACP (Zhang, Y.-M., Marrakchi, H., White, S. W., and Rock, C. O. (2003) J. Lipid Res. 44, 1-10). We tested this hypothesis by mutating two surface residues, Arg-129 and Arg-172, located in a hydrophobic patch adjacent to the active site entrance on beta-ketoacyl-ACP reductase (FabG). Enzymatic analysis showed that the mutant enzymes were compromised in their ability to utilize ACP thioester substrates but were fully active in assays with a substrate analog. Direct binding assays and competitive inhibition experiments showed that the FabG mutant proteins had reduced affinities for ACP. Chemical shift perturbation protein NMR experiments showed that FabG-ACP interactions occurred along the length of ACP helix alpha2 and extended into the adjacent loop-2 region to involve Ile-54. These data confirm a role for the highly conserved electronegative/hydrophobic residues along ACP helix alpha2 in recognizing a constellation of Arg residues embedded in a hydrophobic patch on the surface of its partner enzymes, and reveal a role for the loop-2 region in the conformational change associated with ACP binding. The specific FabG-ACP interactions involve the most conserved ACP residues, which accounts for the ability of ACPs and the type II proteins from different species to function interchangeably.     

Functional characterization of beta-ketoacyl-ACP reductase (FabG) from Plasmodium falciparum

The malaria parasite, Plasmodium falciparum, unlike its human host, utilizes type II fatty acid synthesis, in which steps of fatty acid biosynthesis are catalyzed by independent enzymes. Due to this difference, the enzymes of this pathway are a potential target of newer antimalarials. Here we report the functional characterization of Plasmodium FabG expressed in Escherichia coli. The purified recombinant FabG from P. falciparum is soluble and active. The K(m) of the enzyme for acetoacetyl-CoA was estimated to be 75 microM with a V(max) of 0.0054 micromol/min/ml and a k(cat) value of 0.014s(-1). NADPH exhibited negative cooperativity for its interaction with FabG. We have also modeled P. falciparum FabG using Brassica napus FabG as the template. This model provides a structural rationale for the specificity of FabG towards its cofactor, NADPH.     

Kinetic and nuclear magnetic resonance study of the interaction of NADP+ and NADPH with chicken liver fatty acid synthase

The ionic strength dependence of the second-order rate constant for the association of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and chicken liver fatty acid synthase was determined. This rate constant is 7.2 X 10(7) M-1 s-1 at zero ionic strength and 25 degrees C; the effective charge at the cofactor binding sites is +0.8. The conformations of nicotinamide adenine dinucleotide phosphate (NADP+) and NADPH bound to the beta-ketoacyl and enoyl reductase sites were determined from transferred nuclear Overhauser effect measurements. Covalent modification of the enzyme with pyridoxal 5'-phosphate abolished cofactor binding at the enoyl reductase site; this permitted the cofactor conformations at the beta-ketoacyl and enoyl reductase sites to be distinguished. For NADP+ bound to the enzyme, the conformation of the nicotinamide-ribose bond is anti at the enoyl reductase site and syn at the beta-ketoacyl reductase site; the adenine-ribose bond is anti, and the sugar puckers are C3'-endo. Nicotinamide-adenine base stacking was not detected. Structural models of NADP+ at the beta-ketoacyl and enoyl reductase sites were constructed by using the distances calculated from the observed nuclear Overhauser effects. Because of the overlap of the resonances of several nonaromatic NADPH protons with the resonances of HDO and ribose protons, less extensive structural information was obtained for NADPH bound to the enzyme. However, the conformations of NADPH bound to the two reductases are qualitatively the same as those of NADP+, except that the nicotinamide moiety of NADPH is closer to being fully anti at the enoyl reductase site.     

Crystal structures of Enoyl-ACP reductases I (FabI) and III (FabL) from B. subtilis

Enoyl-[acyl carrier protein] (ACP) reductase (ENR) is a key enzyme in type II fatty acid synthesis that catalyzes the last step in each elongation cycle. Therefore, it has been considered as a target for antibiotics. However, recent studies indicate that some pathogens have more than one ENR; in particular, Bacillus subtilis has two ENRs, FabI and FabL. The crystal structures of the ternary complexes of BsFaBI and BsFabL are found as a homotetramer showing the same overall structure despite a sequence identity of only 24%. The positions of the catalytic dyad of Tyr-(Xaa)₆-Lys in FabL are almost identical to that of FabI, but a detailed structural analysis shows that FabL shares more structural similarities with FabG and other members of the SDR (short-chain alcohol dehydrogenase/reductase) family. The apo FabL structure shows significantly different conformations at the cofactor and the substrate-binding regions, and this resulted in a totally different tetrameric arrangement reflecting the flexibility of these regions in the absence of the cofactor and substrate/inhibitor.     

RhlA converts beta-hydroxyacyl-acyl carrier protein intermediates in fatty acid synthesis to the beta-hydroxydecanoyl-beta-hydroxydecanoate component of rhamnolipids in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the beta-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of beta-hydroxydecanoyl-beta-hydroxydecanoate from two molecules of beta-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily beta-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity in P. aeruginosa increases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for beta-hydroxydecanoyl-ACP. In Escherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.     

Analysis of Streptomyces coelicolor phosphopantetheinyl transferase, AcpS, reveals the basis for relaxed substrate specificity

The transfer of the phosphopantetheine chain from coenzyme A (CoA) to the acyl carrier protein (ACP), a key protein in both fatty acid and polyketide synthesis, is catalyzed by ACP synthase (AcpS). Streptomyces coelicolor AcpS is a doubly promiscuous enzyme capable of activation of ACPs from both fatty acid and polyketide synthesis and catalyzes the transfer of modified CoA substrates. Five crystal structures have been determined, including those of ligand-free AcpS, complexes with CoA and acetyl-CoA, and two of the active site mutants, His110Ala and Asp111Ala. All five structures are trimeric and provide further insight into the mechanism of catalysis, revealing the first detailed structure of a group I active site with the essential magnesium in place. Modeling of ACP binding supported by mutational analysis suggests an explanation for the promiscuity in terms of both ACP partner and modified CoA substrates.     

Structural insight into bioremediation of triphenylmethane dyes by Citrobacter sp. triphenylmethane reductase

Triphenylmethane dyes are aromatic xenobiotic compounds that are widely considered to be one of the main culprits of environmental pollution. Triphenylmethane reductase (TMR) from Citrobacter sp. strain KCTC 18061P was initially isolated and biochemically characterized as an enzyme that catalyzes the reduction of triphenylmethane dyes. Information from the primary amino acid sequence suggests that TMR is a dinucleotide-binding motif-containing enzyme; however, no other functional clues can be derived from sequence analysis. We present the crystal structure of TMR in complex with NADP+ at 2.0-angstroms resolution. Despite limited sequence similarity, the enzyme shows remarkable structural similarity to short-chain dehydrogenase/reductase (SDR) family proteins. Functional assignments revealed that TMR has features of both classic and extended SDR family members and does not contain a conserved active site. Thus, it constitutes a novel class of SDR family proteins. On the basis of simulated molecular docking using the substrate malachite green and the TMR/NADP+ crystal structure, together with site-directed mutagenesis, we have elucidated a potential molecular mechanism for triphenylmethane dye reduction.     

Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by pseudomonads: establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli.

Since Pseudomonas aeruginosa is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various fab mutants from P. aeruginosa with respect to biosynthesis of PHAs and rhamnolipids. All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P. aeruginosa showed decreased PHA accumulation and rhamnolipid production. In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of phaG from Pseudomonas putida and expression of the beta-ketoacyl reductase gene rhlG from P. aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P. putida strongly impaired in PHA production from gluconate. All mutants were complemented by the phaG gene from P. putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E. coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P. putida, only when fatty acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.