The 3-Oxoacyl-(Acyl-Carrier-Protein) Reductase HSD-X1 of Pseudomonas Citronellolis SJTE-3 Catalyzes the Conversion of 17ß-estradiol to Estrone.

BACKGROUND: Pseudomonas citronellolis SJTE-3 can efficiently degrade 17ß-estradiol (E2) and other estrogenic chemicals. However, the enzyme responsible for E2 metabolism within strain SJTE-3 has remained unidentified. OBJECTIVE: Here, a novel 3-oxoacyl-(acyl-carrier protein) (ACP) reductase, HSD-X1 (WP_ 009617962.1), was identified in SJTE-3 and its enzymatic characteristics for the transformation of E2 were investigated. METHODS: Multiple sequence alignment and homology modelling were used to predict the protein structure of HSD-X1. The concentrations of different steroids in the culture of recombinant strains expressing HSD-X1 were determined by high performance liquid chromatography. Additionally, the transcription of hsd-x1 gene was investigated using reverse transcription and quantitative PCR analysis. Heterologous expression and affinity purification were used to obtain recombinant HSD- X1. RESULTS: The transcription of hsd-x1 gene in P. citronellolis SJTE-3 was induced by E2. Multiple sequence alignment (MSA) indicated that HSD-X1 contained the two consensus regions and conserved residues of short-chain dehydrogenase/reductases (SDRs) and 17ß-hydroxysteroid dehydrogenases (17ß-HSDs). Over-expression of hsd-x1 gene allowed the recombinant strain to degrade E2. Recombinant HSD-X1 was purified with a yield of 22.15 mg/L and used NAD+ as its cofactor to catalyze the oxidization of E2 into estrone (E1) while exhibiting a Km value of 0.025 ± 0.044 mM and a Vmax value of 4.92 ± 0.31 mM/min/mg. HSD-X1 could tolerate a wide range of temperature and pH, while the presence of divalent ions exerted little influence on its activity. Further, the transformation efficiency of E2 into E1 was over 98.03% across 15 min. CONCLUSION: Protein HSD-X1 efficiently catalyzed the oxidization of E2 and participated in estrogen degradation by P. citronellolis SJTE-3.

Interaction of ONION2 ketoacyl CoA synthase with ketoacyl CoA reductase of rice.

BACKGROUND: Fatty acid elongases (FAEs), which catalyse elongation reactions of a carbon chain of very-long-chain fatty acids, play an important role in shoot development in rice. The elongation reactions consist of four sequential reactions catalysed by distinct enzymes, which are assumed to form an elongation complex. However, no interacting proteins of ONION1 (ONI1) and ONI2, which are ketoacyl CoA synthase catalyzing the first step and are required for shoot development in rice, are reported. METHODS AND RESULTS: In this study ketoacyl CoA reductase (KCR) that interacts with ONI1 and ONI2 was searched. A database search identified 10 KCR genes in the rice genome. Among the genes, the expression pattern of KCR1 was similar to that of ONI2. Yeast two-hybrid analysis showed interaction of ONI2 with KCR1, which was confirmed by GST pull-down assay. No interacting partner of ONI1 was identified. CONCLUSIONS: Our results suggest that ONI2 and KCR1 form an FAE complex that may play a role in biosynthesizing VLCFAs during shoot development.

Computational characterisation of Toxoplasma gondii FabG (3-oxoacyl-[acyl-carrier-protein] reductase): a combined virtual screening and all-atom molecular dynamics simulation study.

Toxoplasma gondii is an opportunistic obligate parasite, ubiquitous around the globe with seropositivity rates that range from 10% to 90% and infection by the parasite of pregnant women causes pre-natal death of the foetus in most cases and severe neurodegenerative syndromes in some. No vaccine is currently available, and since drug-resistance is common among T. gondii strains, discovering lead compounds for drug design using diverse tactics is necessary. In this study, the sole constituent isoform of an enzymatic 3-oxoacyl-[acyl-carrier-protein] reduction step in an apicoplast-located fatty acid biosynthesis pathway was chosen as a possible drug target. FASII is prokaryotic therefore, targeting it would pose fewer side-effects to human hosts. After a homology 3D modelling of TgFabG, a high-throughput virtual screening of 9867 compounds, the elimination of ligands was carried out by a flexible ligand molecular docking and 200 ns molecular dynamics simulations, with additional DCCM and PC plot analyses. Molecular Dynamics and related post-MD analyses of the top 3 TgFabG binders selected for optimal free binding energies, showed that L2 maintained strong H-bonds with TgFabG and facilitated structural reorientation expected of FabGs, namely an expansion of the Rossmann Fold and a flexible lid capping. The most flexible TgFabG sites were the α7 helix (the flexible lid region) and the ß4-α4 and ß5-α6 loops. For TgFabG-L2, the movements of these regions toward the active site enabled greater ligand stability. Thus, L2 ("Skimmine"; PubChem ID: 320361), was ultimately selected as the optimal candidate for the discovery of lead compounds for rational drug design.Communicated by Ramaswamy H. Sarma.

Sunflower (Helianthus annuus) fatty acid synthase complex: ß-Ketoacyl-[acyl carrier protein] reductase genes.

Fatty acids play many roles in plants, but the function of some key genes involved in fatty acid biosynthesis in plant development are not yet properly understood. Here, we clone two ß-ketoacyl-[ACP] reductase (KAR) genes from sunflower, HaKAR1 and HaKAR2, and characterize their functional roles. The enzymes cloned were the only two copies present in the sunflower genome. Both displayed a high degree of similarity, but their promoters infer different regulation. The two sunflower KAR genes were constitutively expressed in all tissues examined, being maximum in developing cotyledons at the start of oil synthesis. Over-expression of HaKAR1 in E. coli changed the fatty acid composition by promoting the elongation of C16:0 to C18:0 fatty acids. The enzymatic characterization of HaKAR1 revealed similar kinetic parameters to homologues from other oil accumulating species. The results point to a partially functional redundancy between HaKAR1 and HaKAR2. This study clearly revealed that these genes play a prominent role in de novo fatty acids synthesis in sunflower seeds.

Structural comparison of Acinetobacter baumannii ß-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis.

Some Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the ß-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the ß-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure-function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host-pathogen interaction mechanisms and novel antibiotics discovery.

Insights into Acinetobacter baumannii fatty acid synthesis 3-oxoacyl-ACP reductases.

Treatments for 'superbug' infections are the focus for innovative research, as drug resistance threatens human health and medical practices globally. In particular, Acinetobacter baumannii (Ab) infections are repeatedly reported as difficult to treat due to increasing antibiotic resistance. Therefore, there is increasing need to identify novel targets in the development of different antimicrobials. Of particular interest is fatty acid synthesis, vital for the formation of phospholipids, lipopolysaccharides/lipooligosaccharides, and lipoproteins of Gram-negative envelopes. The bacterial type II fatty acid synthesis (FASII) pathway is an attractive target for the development of inhibitors and is particularly favourable due to the differences from mammalian type I fatty acid synthesis. Discrete enzymes in this pathway include two reductase enzymes: 3-oxoacyl-acyl carrier protein (ACP) reductase (FabG) and enoyl-ACP reductase (FabI). Here, we investigate annotated FabG homologs, finding a low-molecular weight 3-oxoacyl-ACP reductase, as the most likely FASII FabG candidate, and high-molecular weight 3-oxoacyl-ACP reductase (HMwFabG), showing differences in structure and coenzyme preference. To date, this is the second bacterial high-molecular weight FabG structurally characterized, following FabG4 from Mycobacterium. We show that ΔAbHMwfabG is impaired for growth in nutrient rich media and pellicle formation. We also modelled a third 3-oxoacyl-ACP reductase, which we annotated as AbSDR. Despite containing residues for catalysis and the ACP coordinating motif, biochemical analyses showed limited activity against an acetoacetyl-CoA substrate in vitro. Inhibitors designed to target FabG proteins and thus prevent fatty acid synthesis may provide a platform for use against multidrug-resistant pathogens including A. baumannii.

Escherichia coli FabG 3-ketoacyl-ACP reductase proteins lacking the assigned catalytic triad residues are active enzymes.

The FabG 3-ketoacyl-acyl carrier protein (ACP) reductase of Escherichia coli has long been thought to be a classical member of the short-chain alcohol dehydrogenase/reductase (SDR) family. FabG catalyzes the essential 3-ketoacyl-ACP reduction step in the FAS II fatty acid synthesis pathway. Site-directed mutagenesis studies of several other SDR enzymes has identified three highly conserved amino acid residues, Ser, Tyr, and Lys, as the catalytic triad. Structural analyses of E. coli FabG suggested the triad S138-Y151-K155 to form a catalytically competent active site. To test this hypothesis, we constructed a series of E. coli FabG mutants and tested their 3-ketoacyl-ACP reductase activities both in vivo and in vitro. Our data show that plasmid-borne FabG mutants, including the double and triple mutants, restored growth of E. coli and Salmonella enterica fabG temperature-sensitive mutant strains under nonpermissive conditions. In vitro assays demonstrated that all of the purified FabG mutant proteins maintained fatty acid synthetic ability, although the activities of the single mutant proteins were 20% to 50% lower than that of wildtype FabG. The S138A, Y151F, and K155A residue substitutions were confirmed by tandem mass spectral sequencing of peptides that spanned all three residues. We conclude that FabG is not a classical short-chain alcohol dehydrogenase/reductase, suggesting that an alternative mode of 3-ketoacyl-ACP reduction awaits discovery.

MoS2-Calix[4]arene Catalyzed Synthesis and Molecular Docking Study of 2,4,5-Trisubstituted Imidazoles As Potent Inhibitors of Mycobacterium tuberculosis.

A MoS2-supported-calix[4]arene (MoS2-CA4) nanocatalyst was used for efficient synthesis of 2,4,5-trisubstituted imidazole derivatives from 1-(4-nitrophenyl)-2-(4-(trifluoromethyl)phenyl)ethane-1,2-dione, aldehydes and ammonium acetate under solvent-free conditions. Reusability of the catalyst up to five cycles without any significant loss in the yields of the product is the unique feature of this heterogeneous solid catalysis. Furthermore, the noteworthy highlights of this method are safe reaction profiles, broad substrate scope, excellent yields, economical, solvent-free, and simple workup conditions. All synthesized compounds were evaluated for their in vitro antitubercular (TB) activity against Mycobacterium tuberculosis (Mtb) H37Rv. Among the screened compounds 3c, 3d, 3f, 3m, and 3r had MIC values of 2.15, 2.78, 5.75, 1.36, and 0.75 µM, respectively, and exhibited more potency than the reference drugs pyrazinamide (MIC: 3.12 µM), ciprofloxacin (MIC: 4.73 µM), and ethambutol (7.61 µM). Besides, potent compounds (3c, 3d, 3f, 3m, and 3r) have been tested for inhibition of MabA (ß-ketoacyl-ACP reductase) enzyme and cytotoxic activity against mammalian Vero cell line. A molecular docking study was carried out on the MabA (PDB ID: 1UZN) enzyme to predict the interactions of the synthesized compounds. The results of the in vitro anti-TB activity and docking study showed that synthesized compounds have a strong anti-TB activity and can be adapted and produced more effectively as a lead compound.

Dual-Localized Enzymatic Components Constitute the Fatty Acid Synthase Systems in Mitochondria and Plastids.

Plant fatty acid biosynthesis occurs in both plastids and mitochondria. Here, we report the identification and characterization of Arabidopsis (Arabidopsis thaliana) genes encoding three enzymes shared between the mitochondria- and plastid-localized type II fatty acid synthase systems (mtFAS and ptFAS, respectively). Two of these enzymes, ß-ketoacyl-acyl carrier protein (ACP) reductase and enoyl-ACP reductase, catalyze two of the reactions that constitute the core four-reaction cycle of the FAS system, which iteratively elongates the acyl chain by two carbon atoms per cycle. The third enzyme, malonyl-coenzyme A:ACP transacylase, catalyzes the reaction that loads the mtFAS system with substrate by malonylating the phosphopantetheinyl cofactor of ACP. GFP fusion experiments revealed that the these enzymes localize to both chloroplasts and mitochondria. This localization was validated by characterization of mutant alleles, which were rescued by transgenes expressing enzyme variants that were retargeted only to plastids or only to mitochondria. The singular retargeting of these proteins to plastids rescued the embryo lethality associated with disruption of the essential ptFAS system, but these rescued plants displayed phenotypes typical of the lack of mtFAS function, including reduced lipoylation of the H subunit of the glycine decarboxylase complex, hyperaccumulation of glycine, and reduced growth. However, these latter traits were reversible in an elevated-CO2 atmosphere, which suppresses mtFAS-associated photorespiration-dependent chemotypes. Sharing enzymatic components between mtFAS and ptFAS systems constrains the evolution of these nonredundant fatty acid biosynthetic machineries.

A novel 3-oxoacyl-ACP reductase (FabG3) is involved in the xanthomonadin biosynthesis of Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, produces a membrane-bound yellow pigment called xanthomonadin to protect against photobiological and peroxidative damage, and uses a quorum-sensing mechanism mediated by the diffusible signal factor (DSF) family signals to regulate virulence factors production. The Xcc gene XCC4003, annotated as Xcc fabG3, is located in the pig cluster, which may be responsible for xanthomonadin synthesis. We report that fabG3 expression restored the growth of the Escherichia coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrated that FabG3 catalyses the reduction of 3-oxoacyl-acyl carrier protein (ACP) intermediates in fatty acid synthetic reactions, although FabG3 had a lower activity than FabG1. Moreover, the fabG3 deletion did not affect growth or fatty acid composition. These results indicate that Xcc fabG3 encodes a 3-oxoacyl-ACP reductase, but is not essential for growth or fatty acid synthesis. However, the Xcc fabG3 knock-out mutant abolished xanthomonadin production, which could be only restored by wild-type fabG3, but not by other 3-oxoacyl-ACP reductase-encoding genes, indicating that Xcc FabG3 is specifically involved in xanthomonadin biosynthesis. Additionally, our study also shows that the Xcc fabG3-disrupted mutant affects Xcc virulence in host plants.

Mutation in the putative ketoacyl-ACP reductase CaKR1 induces loss of pungency in Capsicum.

KEY MESSAGE: A putative ketoacyl-ACP reductase (CaKR1) that was not previously known to be associated with pungency of Capsicum was identified from map-based cloning and functional characterization. The pungency of chili pepper fruits is due to the presence of capsaicinoids, which are synthesized through the convergence of the phenylpropanoid and branched-chain fatty acid pathways. The extensive, global use of pungent and non-pungent peppers underlines the importance of understanding the genetic mechanism underlying capsaicinoid biosynthesis for breeding pepper cultivars. Although Capsicum is one of the earliest domesticated plant genera, the only reported genetic causes of its loss of pungency are mutations in acyltransferase (Pun1) and putative aminotransferase (pAMT). In this study, a single recessive gene responsible for the non-pungency of pepper No.3341 (C. chinense) was identified on chromosome 10 using an F2 population derived from a cross between Habanero and No.3341. Five candidate genes were identified in the target region, within a distance of 220 kb. A candidate gene, a putative ketoacyl-ACP reductase (CaKR1), of No.3341 had an insertion of a 4.5-kb transposable element (TE) sequence in the first intron, resulting in the production of a truncated transcript missing the region coding the catalytic domain. Virus-induced gene silencing of CaKR1 in pungent peppers resulted in the decreased accumulation of capsaicinoids, a phenotype consistent with No.3341. Moreover, GC-MS analysis of 8-methyl-6-nonenoic acid, which is predicted to be synthesized during the elongation cycle of branched-chain fatty acid biosynthesis, revealed that its deficiency in No.3341. Genetic, genomic, transcriptional, silencing, and biochemical precursor analyses performed in combination provide a solid ground for the conclusion that CaKR1 is involved in capsaicinoid biosynthesis and that its disruption results in a loss of pungency.

Stereoselective Bioreduction of Ethyl 3-Oxo-3-(2-Thienyl) Propanoate Using the Short-Chain Dehydrogenase/Reductase ChKRED12.

Ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate((S)-HEES)acts as a key chiral intermediate for the blockbuster antidepressant drug duloxetine, which canbe achieved viathe stereoselective bioreduction ofethyl 3-oxo-3-(2-thienyl) propanoate (KEES) that containsa 3-oxoacyl structure.The sequences of the short-chain dehydrogenase/reductases from Chryseobacterium sp. CA49 were analyzed, and the putative3-oxoacyl-acyl-carrier-protein reductase, ChKRED12, was able to stereoselectivelycatalyze theNADPH-dependent reduction to produce (S)-HEES.The reductase activity of ChKRED12 towardsothersubstrates with 3-oxoacyl structure were confirmed with excellent stereoselectivity (>99% enantiomeric excess) in most cases. When coupled with a cofactor recycling system using glucose dehydrogenase, the ChKRED12 was able to catalyze the complete conversion of 100 g/l KEES within 12h, yielding the enantiopure product with >99% ee, showing a remarkable potential to produce (S)-HEES.

One 3-oxoacyl-(acyl-Carrier-protein) reductase functions as 17ß-hydroxysteroid dehydrogenase in the estrogen-degrading Pseudomonas putida SJTE-1.

Pseudomonas putida SJTE-1 can utilize 17ß-estradiol (E2) as its carbon source, while the enzymes for E2 transformation in this strain is still unclear. 17ß-hydroxysteroid dehydrogenases (17ß-HSD) can catalyze the reduction/oxidation at C17 site of steroid hormone specifically, critical for steroid transformation. Here a novel 3-oxoacyl-(acyl-carrier protein) (ACP) reductase (ANI02794.1) was identified as it could bß-estradiol, and was proved to be capable of functioning as 17ß-HSD. Sequences alignment showed it contained the two consensus regions and the conserved residues of short-chain dehydrogenase/reductase (SDR). Its encoding gene was cloned and over-expressed in Escherichia coli BL21(DE3) strain, and the recombinant protein was purified by the metal-ion affinity chromatography with the yield of 18 mg/L culture. HPLC (High Performance Liquid Chromatography) detection showed this enzyme could convert 17ß-estradiol into estrone using NAD+ as cofactor. Its Km value was 0.082 mM and its Vmax value was 0.81 mM/s; its transformation efficiency of 17ß-estradiol into estrone was over 96.6% in five minutes. Its optimal temperature was 37 °C and optimal was pH 9.0; the divalent ions had different effects on the enzymatic activity. In conclusion, this 3-oxoacyl-ACP reductase functioned as 17ß-HSD in P. putida SJTE-1 and played important role in its estrogen metabolism.

Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis ß-ketoacyl-acyl carrier protein reductase MabA.

In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of ß-ketoacyl-ACP substrates to ß-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP+-bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP+-bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

Plasmid AZOBR_p1-borne fabG gene for putative 3-oxoacyl-[acyl-carrier protein] reductase is essential for proper assembly and work of the dual flagellar system in the alphaproteobacterium Azospirillum brasilense Sp245.

Azospirillum brasilense can swim and swarm owing to the activity of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf), respectively. Experimental data on the regulation of the Fla and Laf assembly in azospirilla are scarce. Here, the coding sequence (CDS) AZOBR_p1160043 (fabG1) for a putative 3-oxoacyl-[acyl-carrier protein (ACP)] reductase was found essential for the construction of both types of flagella. In an immotile leaky Fla- Laf- fabG1::Omegon-Km mutant, Sp245.1610, defects in flagellation and motility were fully complemented by expressing the CDS AZOBR_p1160043 from plasmid pRK415. When pRK415 with the cloned CDS AZOBR_p1160045 (fliC) for a putative 65.2 kDa Sp245 Fla flagellin was transferred into the Sp245.1610 cells, the bacteria also became able to assemble a motile single flagellum. Some cells, however, had unusual swimming behavior, probably because of the side location of the organelle. Although the assembly of Laf was not restored in Sp245.1610 (pRK415-p1160045), this strain was somewhat capable of swarming motility. We propose that the putative 3-oxoacyl-[ACP] reductase encoded by the CDS AZOBR_p1160043 plays a role in correct flagellar location in the cell envelope and (or) in flagellar modification(s), which are also required for the inducible construction of Laf and for proper swimming and swarming motility of A. brasilense Sp245.

A QM/MM study of the reaction mechanism of human ß-ketoacyl reductase.

Human fatty acid synthase (hFAS) is a multifunctional enzyme involved in a wide diversity of biological functions. For instance, it is a precursor of phospholipids and other complex processes such as the de novo synthesis of long chain fatty acid. Human FAS is also a component of biological membranes and it is implicated in the overexpression of several types of cancers. In this work, we describe the catalytic mechanism of ß-ketoreductase (KR), which is a catalytic domain of the hFAS enzyme that catalyzes the reduction of ß-ketoacyl to ß-hydroxyacyl with the concomitant oxidation of the NADPH cofactor. The catalysis by KR is an intermediate step in the cycle of reactions that elongate the substrate's carbon chain until the final product is obtained. We study and propose the catalytic mechanism of the KR domain determined using the hybrid QM/MM methodology, at the ONIOM(B3LYP/6-311+G(2d,2p):AMBER) level of theory. The results indicate that the reaction mechanism occurs in two stages: (i) nucleophilic attack by a NADPH hydride to the ß-carbon of the substrate, together with an asynchronous deprotonation of the Tyr2034 by the oxygen of the ß-alkoxide to hold the final alcohol product; and (ii) an asynchronous deprotonation of the hydroxyl in the NADP+'s ribose by Tyr2034, and of the Lys1995 by the resulting alkoxide in the former ribose to restore the protonation state of Tyr2034. The reduction step occurs with a Gibbs energy barrier of 11.7 kcal mol-1 and a Gibbs reaction energy of -10.6 kcal mol-1. These results have provided an understanding of the catalytic mechanism of the KR hFAS domain, a piece of the heavy hFAS biosynthetic machinery.

Proteomic analysis of soybean root exposed to varying sizes of silver nanoparticles under flooding stress.

UNLABELLED: Silver nanoparticles (Ag-NPs) are excessively used as antibacterial agents; however, environmental interaction specifically with the plants remain uncertain. To study the size-dependent effects of Ag-NPs on soybean under flooding, a proteomic technique was used. Morphological analysis revealed that treatment with Ag-NPs of 15nm promoted soybean growth under flooding compared to 2 and 50-80nm. A total of 228 common proteins that significantly changed in abundance under flooding without and with Ag-NPs of 2, 15, and 50-80nm. Under varying sizes of Ag-NPs, number of protein synthesis related proteins decreased compared to flooding while number of amino acid synthesis related proteins were increased under Ag-NPs of 15nm. Hierarchical clustering identified the ribosomal proteins that increased under Ag-NPs of 15nm while decreased under other sizes. In silico protein-protein interaction indicated the beta ketoacyl reducatse 1 as the most interacted protein under Ag-NPs of 15nm while least interacted under other sizes. The beta ketoacyl reductase 1 was up-regulated under Ag-NPs of 15nm while its enzyme activity was decreased. These results suggest that the different sizes of Ag-NPs might affect the soybean growth under flooding by regulating the proteins related to amino acid synthesis and wax formation. BIOLOGICAL SIGNIFICANCE: This study highlighted the response of soybean proteins towards varying sizes of Ag NPs under flooding stress using gel-free proteomic technique. The Ag NPs of 15nm improved the length of root including hypocotyl of soybean. The proteins related to protein metabolism, cell division/organization, and amino acid metabolism were differentially changed under the varying sizes of Ag NPs. The protein synthesis-related proteins were decreased while amino acid metabolism-related proteins were increased under varying sizes of Ag NPs. The ribosomal proteins were increased under Ag NPs of 15nm. The beta ketoacyl reductase 1 was identified as the most interacted protein under varying sizes of Ag NPs. The mRNA expression level of beta ketoacyl reductase was up-regulated under Ag NPs of 15nm while its activity was decreased. These results suggest that the Ag NPs of 15nm improved the soybean growth under flooding stress by increasing the proteins related to amino acid synthesis and waxes formation.

Sinorhizobium meliloti Functionally Replaces 3-Oxoacyl-Acyl Carrier Protein Reductase (FabG) by Overexpressing NodG During Fatty Acid Synthesis.

In Sinorhizobium meliloti, the nodG gene is located in the nodFEG operon of the symbiotic plasmid. Although strong sequence similarity (53% amino acid identities) between S. meliloti NodG and Escherichia coli FabG was reported in 1992, it has not been determined whether S. meliloti NodG plays a role in fatty acid synthesis. We report that expression of S. meliloti NodG restores the growth of the E. coli fabG temperature-sensitive mutant CL104 under nonpermissive conditions. Using in vitro assays, we demonstrated that NodG is able to catalyze the reduction of the 3-oxoacyl-ACP intermediates in E. coli fatty acid synthetic reaction. Moreover, although deletion of the S. meliloti nodG gene does not cause any growth defects, upon overexpression of nodG from a plasmid, the S. meliloti fabG gene encoding the canonical 3-oxoacyl-ACP reductase (OAR) can be disrupted without any effects on growth or fatty acid composition. This indicates that S. meliloti nodG encodes an OAR and can play a role in fatty acid synthesis when expressed at sufficiently high levels. Thus, a bacterium can simultaneously possess two or more OARs that can play a role in fatty acid synthesis. Our data also showed that, although SmnodG increases alfalfa nodulation efficiency, it is not essential for alfalfa nodulation.

Dissecting the Structural Elements for the Activation of ß-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae.

UNLABELLED: ß-Ketoacyl-(acyl carrier protein) reductase (FabG) catalyzes the key reductive reaction in the elongation cycle of fatty acid synthesis (FAS), which is a vital metabolic pathway in bacteria and a promising target for new antibiotic development. The activation of the enzyme is usually linked to the formation of a catalytic triad and cofactor binding, and crystal structures of FabG from different organisms have been captured in either the active or inactive conformation. However, the structural elements which enable activation of FabG require further exploration. Here we report the findings of structural, enzymatic, and binding studies of the FabG protein found in the causative agent of cholera, Vibrio cholerae (vcFabG). vcFabG exists predominantly as a dimer in solution and is able to self-associate to form tetramers, which is the state seen in the crystal structure. The formation of the tetramer may be promoted by the presence of the cofactor NADP(H). The transition between the dimeric and tetrameric states of vcFabG is related to changes in the conformations of the α4/α5 helices on the dimer-dimer interface. Two glycine residues adjacent to the dimer interface (G92 and G141) are identified to be the hinge for the conformational changes, while the catalytic tyrosine (Y155) and a glutamine residue that forms hydrogen bonds to both loop ß4-α4 and loop ß5-α5 (Q152) stabilize the active conformation. The functions of the aforementioned residues were confirmed by binding and enzymatic assays for the corresponding mutants. IMPORTANCE: This paper describes the results of structural, enzymatic, and binding studies of FabG from Vibrio cholerae (vcFabG). In this work, we dissected the structural elements responsible for the activation of vcFabG. The structural information provided here is essential for the development of antibiotics specifically targeting bacterial FabG, especially for the multidrug-resistant strains of V. cholerae.

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.