Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus

A 5.2 kb region from the oleandomycin gene cluster in Streptomyces antibioticus located between the oleandomycin polyketide synthase gene and sugar biosynthetic genes was cloned. Sequence analysis revealed the presence of three open reading frames (designated oleI , oleN2 and oleR ). The oleI gene product resembled glycosyltransferases involved in macrolide inactivation including the oleD product, a previously described glycosyltransferase from S. antibioticus . The oleN2 gene product showed similarities with different aminotransferases involved in the biosynthesis of 6-deoxyhexoses. The oleR gene product was similar to several glucosidases from different origins. The oleI , oleR and oleD genes were expressed in Streptomyces lividans . OleI and OleD intracellular proteins were partially purified by affinity chromatography in an UDP-glucuronic acid agarose column and OleR was detected as a major band from the culture supernatant. OleI and OleD showed oleandomycin glycosylating activity but they differ in the pattern of substrate specificity: OleI being much more specific for oleandomycin. OleR showed glycosidase activity converting glycosylated oleandomycin into active oleandomycin. A model is proposed integrating these and previously reported results for intracellular inactivation, secretion and extracellular reactivation of oleandomycin.     

Functional characterization of an NADPH dependent 2-alkyl-3-ketoalkanoic acid reductase involved in olefin biosynthesis in Stenotrophomonas maltophilia

OleD is shown to play a key reductive role in the generation of alkenes (olefins) from acyl thioesters in Stenotrophomonas maltophilia. The gene coding for OleD clusters with three other genes, oleABC, and all appear to be transcribed in the same direction as an operon in various olefin producing bacteria. In this study, a series of substrates varying in chain length and stereochemistry were synthesized and used to elucidate the functional role and substrate specificity of OleD. We demonstrated that OleD, which is an NADP(H) dependent reductase, is a homodimer which catalyzes the reversible stereospecific reduction of 2-alkyl-3-ketoalkanoic acids. Maximal catalytic efficiency was observed with syn-2-decyl-3-hydroxytetradecanoic acid, with a k(cat)/K(m) 5- and 8-fold higher than for syn-2-octyl-3-hydroxydodecanoic acid and syn-2-hexyl-3-hydroxydecanoic acid, respectively. OleD activity was not observed with syn-2-butyl-3-hydroxyoctanoic acid and compounds lacking a 2-alkyl group such as 3-ketodecanoic and 3-hydroxydecanoic acids, suggesting the necessity of the 2-alkyl chain for enzyme recognition and catalysis. Using diastereomeric pairs of substrates and 4 enantiopure isomers of 2-hexyl-3-hydroxydecanoic acid of known stereochemistry, OleD was shown to have a marked stereochemical preference for the (2R,3S)-isomer. Finally, experiments involving OleA and OleD demonstrate the first 3 steps and stereochemical course in olefin formation from acyl thioesters; condensation to form a 2-alkyl-3-ketoacyl thioester, subsequent thioester hydrolysis, and ketone reduction.     

Glycosylation of various flavonoids by recombinant oleandomycin glycosyltransferase from <Emphasis Type="Italic">Streptomyces antibioticus</Emphasis> in batch and repeated batch modes

An oleandomycin glycosyltransferase (OleD GT) gene from Streptomyces antibioticus was functionally expressed in Escherichia coli BL21 (DE3) with various molecular chaperones. The purified recombinant OleD GT catalyzed glycosylation of various flavonoids: apigenin, chrysin, daidzein, genistein, kaempferol, luteolin, 4-methylumbelliferone, naringenin, quercetin and resveratrol with UDP–glucose. 4.6 μg OleD GT was readily immobilized onto 1 mg hybrid nanoparticles of Fe₃O₄/silica/NiO on the basis of the affinity between His-tag and NiO nanoparticles with retention of 90% activity. In batch reaction, more than 90% naringenin (20 μM) was converted to its glycoside in 5 h. The immobilized OleD GT was efficiently reused for seven times whilst maintaining >60% of the residual activity in repeated glycosylation of naringenin.     

Identification of Redox Partners and Development of a Novel Chimeric Bacterial Nitric Oxide Synthase for Structure Activity Analyses

Production of nitric oxide (NO) by nitric oxide synthase (NOS) requires electrons to reduce the heme iron for substrate oxidation. Both FAD and FMN flavin groups mediate the transfer of NADPH derived electrons to NOS. Unlike mammalian NOS that contain both FAD and FMN binding domains within a single polypeptide chain, bacterial NOS is only composed of an oxygenase domain and must rely on separate redox partners for electron transfer and subsequent activity. Here, we report on the native redox partners for Bacillus subtilis NOS (bsNOS) and a novel chimera that promotes bsNOS activity. By identifying and characterizing native redox partners, we were also able to establish a robust enzyme assay for measuring bsNOS activity and inhibition. This assay was used to evaluate a series of established NOS inhibitors. Using the new assay for screening small molecules led to the identification of several potent inhibitors for which bsNOS-inhibitor crystal structures were determined. In addition to characterizing potent bsNOS inhibitors, substrate binding was also analyzed using isothermal titration calorimetry giving the first detailed thermodynamic analysis of substrate binding to NOS.     

Nature-inspired engineering of an artificial ligase enzyme by domain fusion

The function of most proteins is accomplished through the interplay of two or more protein domains and fine-tuned by natural evolution. In contrast, artificial enzymes have often been engineered from a single domain scaffold and frequently have lower catalytic activity than natural enzymes. We previously generated an artificial enzyme that catalyzed an RNA ligation by >2 million-fold but was likely limited in its activity by low substrate affinity. Inspired by nature''s concept of domain fusion, we fused the artificial enzyme to a series of protein domains known to bind nucleic acids with the goal of improving its catalytic activity. The effect of the fused domains on catalytic activity varied greatly, yielding severalfold increases but also reductions caused by domains that previously enhanced nucleic acid binding in other protein engineering projects. The combination of the two better performing binding domains improved the activity of the parental ligase by more than an order of magnitude. These results demonstrate for the first time that nature''s successful evolutionary mechanism of domain fusion can also improve an unevolved primordial-like protein whose structure and function had just been created in the test tube. The generation of multi-domain proteins might therefore be an ancient evolutionary process.     

Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [³H]arginine to [³H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor N^G-methyl- -arginine ( -NMA), but not the arginase inhibitors -valine and -ornithine. NOS activity was predominantly cytosolic, and was characterised by a K_m for arginine of 19.05 μM and a V_max of 2.96 pmol/min per μg protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.     

Reconstitution in vivo of penicillin G acylase activity from separately expressed subunits.

Penicillin G acylase from Escherichia coli ATCC11105 is synthesized as a precursor polypeptide with a signal sequence for secretion into the periplasm and an endopeptide separating two subunit domains. Proteolytic processing leads to mature, heterodimeric penicillin G acylase. We have shown that the alpha- and beta-subunits of the enzyme, which have no detectable enzymatic activity on their own, can reconstitute enzyme activity when their genes are put into an E. coli host on separate plasmids. Activity is reconstituted in the cytoplasm whereas normally processing and formation of the active heterodimer occurs in the periplasm. Enzyme activity can reach levels close to wild type in the strain used. The activity recovered from a combination of alpha-subunit linked to a 54-amino-acid endopeptide and beta-subunit was lower than with the subunits alone.     

Studies on regulatory functions of malic enzymes. IV. Effects of sulfhydryl group modification on the catalytic function of NAD-linked malic enzyme from Escherichia coli.

Reactions catalyzed by NAD-linked malic enzyme from Escherichia coli were investigated. In addition to L-malate oxidative decarboxylase activity (Activity 1) and oxaloacetate decarboxylase activity (Activity 2), the enzyme exhibited oxaloacetate reductase activity (Activity 3) and pyruvate reductase activity (Activity 4). Optimum pH's for Activities 3 and 4 were 4.0 and 5.0, and their specific activities were 1.7 and 0.07, respectively. Upon reaction with N-ethylmaleimide (NEM), Activity 1 decreased following pseudo-first order kinetics. Activity 2 decreased in parallel with Activity 1, while Activities 3 and 4 were about ten-fold enhanced by NEM modification. Modification of one or two sulfhydryl groups per enzyme subunit caused an alteration of the activities. Tartronate, a substrate analog, NAD+, and Mn2+ protected the enzyme against the modification. The Km values for the substrates and coenzymes were not significantly affected by NEM modification. Similarly, other sulfhydryl reagents such as p-hydroxymercuribenzoate (PMB), 5,5'-dithiobis(2-nitrobenzoate) (DTNB), and iodoacetate inhibited the decarboxylase activities and activated the reductase activities to various extents. Modification of the enzyme with PMB or DTNB was reversed by the addition of a sulfhydryl compound such as dithiothreitol or 2-mercaptoethanol. Based on the above results, the mechanism of the alteration of enzyme activities by sulfhydryl group modification is discussed.     

Expression of active domains of a human folate-dependent trifunctional enzyme in Escherichia coli

The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase was engineered to contain a prokaryotic ribosome binding site and was expressed under the bacteriophage T7 RNA polymerase promoter in Escherichia coli. Site-directed mutagenesis was used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase (D/C) domain as amino acid residues 1-301, and the synthetase (Syn) domain as residues 304-935. Both domains formed active enzymes thereby demonstrating their ability to fold independently. The full-length enzyme, D/C and Syn domains were expressed at levels 4-, 55- and 3-fold higher than the specific activities found in liver. Additional mutagenesis and independent expression of domains further defined the interdomain region to include amino acids 292-310. The D/C domain was purified to homogeneity by a single affinity chromatographic step, and the full-length protein in a two-step procedure. The kinetic properties of the D/C domain appear unaltered from those of the trifunctional enzyme.     

汉族人群NOS3 A-922G、NOS3 T-786C 与NOS3 G894T SNP的等位基因及其组合分布与高血压的相关性

为了分析汉族人群一氧化氮合酶基因NOS3 A-922G、NOS3 T-786C 与NOS3 G894T单核苷酸多态性（single nucleotide polymorphism,SNP）的等位基因及其组合分布与高血压病的相关性,选取无亲缘关系的高血压病人192例(男97例,女95例)以及无亲缘关系的健康个体122例（男76例,女46例）为对照组,提取静脉血白细胞基因组DNA,采用等位基因特异性引物PCR技术检测NOS3 A-922G、NOS3 T-786C 与NOS3 G894T 3个位点的基因型。其结果显示：高血压病组与对照组NOS3 G894T、NOS3A-922G及NOS3 T-786C各等位基因型及其基因单倍型频率比较无显著性差异(P>0.05)。男、女性别分层研究：无论男亚组还是女亚组均未发现NOS3 A-922G、NOS3 T-786C 与NOS3 G894T各个位点SNP与高血压病有相关性。等位基因组合分布研究发现NOS3 G894G +A-922G+T-786T组合基因型总体频率分布在高血压病组与正常对照组之间有显著性差异(P<0.05,χ2= 4.5944)。男、女性别分层研究：男亚组上述3个位点SNP的各个组合基因型分布频率在高血压病组与正常对照组之间无显著性差异(P>0.05);女亚组中携带NOS3 G894G+A-922G+T-786C 的组合基因型分布频率在高血压病组与正常对照组之间有显著性差异(P<0.01,χ2=8.502)。研究发现,在中国汉族人群中NOS3A–922 G、NOS3 T-786C 与NOS3 G894T SNP与高血压病无明确的相关性,且无性别差异。组合分布研究发现,NOS3 G894G+A-922G+T-786C 的组合基因型分布频率在高血压病女性亚组较健康女性亚组明显减低,提示携带该组合基因型女性人群可能不易患高血压病。     

Anomalous Increase in Nitric Oxide Synthase Activity by Certain Nitric Oxide-Generating Compounds in Intact Neuronal Cells

Abstract: It has been shown that nitric oxide (NO) regulates NO synthase (NOS) activity through negative feedback in cytosolic enzyme preparations in various cell types. We compared the effects of the NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) on NOS activity in intact neuroblastoma N1E-115 cells and in the cytosol obtained from the same cells. Enzyme activity was measured by the conversion of l -[³H]arginine into l -[³H]citrulline. At concentrations that elicit almost complete inhibition of NOS activity in cytosolic enzyme preparations of these cells, SIN-1 and SNP did not cause significant attenuation of enzyme activity measured at 45 min in intact cells. It is surprising that SIN-1 and SNP markedly stimulated l -[³H]citrulline formation in a time- and concentration-dependent manner when cells were incubated with the compounds for >1.5 h. Neither inhibitory nor stimulatory effects of SNAP on NOS were observed in intact N1E-115 cells. This is in contrast to the inhibitory effects of SNAP in cytosolic preparations of the enzyme. The increased NOS activity by SIN-1 or SNP in intact cells was dependent on the presence of extracellular Ca²⁺, suggesting that it might be due to increased Ca²⁺ influx. On the other hand, measurements of the activity of lactate dehydrogenase showed that there was no generalized increase in cell permeability in response to SIN-1 or SNP. There was no agreement in the rank order of potencies of these compounds in activating guanylate cyclase and in affecting NOS activity, both in broken-cell preparations and in intact cells. Thus, modulation of NOS activity by NO-releasing compounds is not dependent on cyclic GMP formation and might not be related in a simple fashion to NO generation. Alternatively, activation of guanylate cyclase and stimulation of NOS activity might require different redox species of NO. Our present findings might be of clinical relevance in relation to long-term use of NO-generating compounds as therapeutic agents.     

Nitric oxide contributes to the spinal nociceptive processing

NADPH-diaphorase-containing neurons (it is supposed that this enzyme is a form of NO-synthase, NOS) were histochemically identified in the spinal cord of rats. In another set of the experiments, we identified neurons -sources of spinothalamic and spinomesencephalic pathways - by their retrograde labelling with Fluoro-Gold (FG) injected into the thalamus and periaqueductal gray substance. It was shown that NOS-containing spinal cells are, as a rule, propriospinal intersegmental or intrasegmental interneurons. We discuss the possible involvement of these cells in the “inhibition-of-inhibition” processes and in potentiation of the synaptic transmission in spinal neurons under conditions of the development of tonic or chronic pain. In rats, the number of NOS-containing neurons, which are also retrogradely labelled with FG after dye injection into the thalamus and midbrain, Is rather limited.     

Nitric oxide synthase II in rat skeletal muscles

Constitutive expression of nitric oxide synthase (NOS) II was found in rat hindlimb muscles by immunohistochemistry and western blotting during development from embryonic day 21 to the adult stage of 75 days. The immunohistochemical NOS II expression pattern was related to the physiological metabolic fibre types SO (slow-oxidative), FOG I, II (fast-oxidative glycolytic; I more glycolytic, II more oxidative) and FG (fast-glycolytic) and to the myosin-based fibre types I and IIA, IIB (IIX not separated) identified in serial sections by enzyme histochemistry and immunohistochemistry. In adult muscles only the small population of FOG II fibres, which is a part of both IIA and IIB fibre population, showed NOS II immunoreactivity. This is the reason that only weak NOS II expression in adult hindlimb muscles has been detected by western blotting. Hindlimb muscles of embryonic, neonatal and young rats of 8 days expressed more NOS II as compared with adult rat hindlimb muscles. This can be explained by the findings that before the age of 21 days fast fibres were metabolically undifferentiated, all of them were NOS II positive and contribute to the NOS II expression of the muscle. In muscles of diabetic rats the NOS II expression was elevated indicating an inhibition of glucose uptake into the muscle fibres of diabetic muscles. Our findings suggest that the NOS II may be designated both as constitutive and inducible.     

Construction and hyperproductivity of engineered strain QE79 bearing recombinant plasmid containing penicillin G acylase gene from Escherichia coli strain AS1.76

Summary An engineered strain QE79 bearing a recombinant plasmid containing the penicillin G acylase gene from E.coli strain AS1.76 was constructed. Formation conditions of the penicillin G acylase were studied. The activity of the enzyme reached over 200 units per 100ml of the culture when the strain QE79 was grown in the medium consisting of mineral salts with supplementary glucose or sucrose at 28°C for 32 hr on shaker. The productivity of the engineered strain QE79 was nearly nine times higher than that of the original strain.     

Functionality of the seventh and eighth transmembrane domains of acyl-coenzyme A:cholesterol acyltransferase 1

Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme in the endoplasmic reticulum. ACAT1 is a homotetrameric protein and contains nine transmembrane domains (TMDs). His460 is a key active residue and is located within TMD7. Human ACAT1 has seven free Cys, but the recombinant ACAT1 devoid of free Cys retains full enzyme activity. To further probe the functionality of TMD7 (amino acids 446-460) and TMD8 (amino acids 466-481), we used a parental ACAT1 devoid of free Cys as the template to perform Cys-scanning mutagenesis within these regions. Each of the single Cys mutants was expressed in Chinese hamster ovary (CHO) cell line AC29 lacking endogenous ACAT1. We measured the effect of single Cys substitution on enzyme activity and used the Cu(1,10-phenanthroline)2SO4-mediated disulfide cross-linking method to probe possible interactions of engineered Cys between the two identical subunits. The results show that several residues in one subunit closely interact with the same residues in the other subunit; mutating these residues to Cys does not lead to large loss in enzyme activity. Helical wheel analysis suggests that these residues are located at one side of the coil. In contrast, mutating residues F453, A457, or H460 to Cys causes large loss in enzyme activity; the latter residues are located at the opposite side of the coil. A similar arrangement is found for residues in TMD8. Thus, helical coils in TMD7 and TMD8 have two distinct functional sides: one side is involved in substrate-binding/catalysis, while the other side is involved in subunit interaction.     

GTP Cyclohydrolase I: Purification, Characterization, and Effects of Inhibition on Nitric Oxide Synthase in Nocardia Species

GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH₄), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a K_m for GTP of 6.5 μM. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56°C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH₄ inhibited enzyme activity by 25% at a concentration of 100 μM. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a K_i of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO₂⁻ plus NO₃⁻. Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH₄.     

Ultrathin nucleoporin phenylalanine–glycine repeat films and their interaction with nuclear transport receptors

Nuclear pore complexes (NPCs) are highly selective gates that mediate the exchange of all proteins and nucleic acids between the cytoplasm and the nucleus. Their selectivity relies on a supramolecular assembly of natively unfolded nucleoporin domains containing phenylalanine–glycine (FG)‐rich repeats (FG repeat domains), in a way that is at present poorly understood. We have developed ultrathin FG domain films that reproduce the mode of attachment and the density of FG repeats in NPCs, and that exhibit a thickness that corresponds to the nanoscopic dimensions of the native permeability barrier. By using a combination of biophysical characterization techniques, we quantified the binding of nuclear transport receptors (NTRs) to such FG domain films and analysed how this binding affects the swelling behaviour and mechanical properties of the films. The results extend our understanding of the interaction of FG domain assemblies with NTRs and contribute important information to refine the model of transport across the permeability barrier.     

Metabolic engineering of type II methanotroph,Methylosinus trichosporium OB3b,for production of 3-hydroxypropionic acid from methane via a malonyl-CoA reductase-dependent pathway

We engineered a type II methanotroph, Methylosinus trichosporium OB3b, for 3-hydroxypropionic acid (3HP) production by reconstructing malonyl-CoA pathway through heterologous expression of Chloroflexus aurantiacus malonyl-CoA reductase (MCR), a bifunctional enzyme. Two strategies were designed and implemented to increase the malonyl-CoA pool and thus, increase in 3HP production. First, we engineered the supply of malonyl-CoA precursors by overexpressing endogenous acetyl-CoA carboxylase (ACC), substantially enhancing the production of 3HP. Overexpression of biotin protein ligase (BPL) and malic enzyme (NADP⁺-ME) led to a ∼22.7% and ∼34.5% increase, respectively, in 3HP titer in ACC-overexpressing cells. Also, the acetyl-CoA carboxylation bypass route was reconstructed to improve 3HP productivity. Co-expression of methylmalonyl-CoA carboxyltransferase (MMC) of Propionibacterium freudenreichii and phosphoenolpyruvate carboxylase (PEPC), which provides the MMC precursor, further improved the 3HP titer. The highest 3HP production of 49 mg/L in the OB3b-MCRMP strain overexpressing MCR, MMC and PEPC resulted in a 2.4-fold improvement of titer compared with that in the only MCR-overexpressing strain. Finally, we could obtain 60.59 mg/L of 3HP in 42 h using the OB3b-MCRMP strain through bioreactor operation, with a 6.36-fold increase of volumetric productivity compared than that in the flask cultures. This work demonstrates metabolic engineering of type II methanotrophs, opening the door for using type II methanotrophs as cell factories for biochemical production along with mitigation of greenhouse gases.     

Amino-Terminal Analysis of Tryptophan Hydroxylase: Protein Kinase Phosphorylation Occurs at Serine-58

Abstract: Tryptophan hydroxylase (TPH) catalyzes the rate-limiting and committed step in serotonin biosynthesis. Within this enzyme, two distinct domains have been hypothesized to exist, an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. In the present experiments, the functional boundary between the putative domains was defined using deletion muta-genesis. A full-length cDNA clone for rabbit TPH was engineered for expression in bacteria. Five amino-terminal deletions were constructed using PCR, i.e., NΔ50, NΔ60, NΔ90, NΔ106, and NΔ116 (referring to the number of amino acids deleted from the amino terminus). Enzymatic activity was determined for each mutant after expression in bacteria. Whereas deletion of 116 amino acids (NΔ116) abolished enzyme activity, all of the other amino-terminal deletions exhibited increased specific activity relative to the recombinant wild-type TPH. The ability of the cyclic AMP-dependent protein kinase (PKA) to phosphorylate members of the deletion series was also examined. Deletion of the first 60 amino-terminal residues abolished the ability of the enzyme to serve as a substrate for PKA, yet the native and NΔ50 enzymes were phosphorylated. Moreover, a serine-58 point mutant (S58A) was not phosphorylated by PKA. In conclusion, the first 106 amino acids comprise a regulatory domain that is phosphorylated by PKA at serine-58. In addition, the boundary between regulatory and catalytic domains is analogous to the domain structure observed for the related enzyme tyrosine hydroxylase.     

New aspects concerning to the characterization and the relationship with the immune response in vivo of the spiny lobster Panulirus argus nitric oxide synthase

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In a previous study, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide (LPS) in vitro. In the present work, lobster hemocytes and gills exposed to Escherichia coli O55:B5 LPS showed an increase in both NOS activity and NOS gene expression in vivo. This response was dose and time dependent. The 3D NOS structure was predicted by comparative modeling showing the oxygenase and reductase domains. These domains contain the conserved binding motifs of NOS already found in a variety of organisms. The 3D structure prediction analysis allowed the selection of a fragment of 666bp that was cloned and subsequently expressed in E. coli BL21, in which a recombinant product of around 31KDa was obtained. Hyperimmune serum obtained from immunized rabbits was tested and employed to specifically detect the recombinant polypeptide or the endogenous NOS from lobster hemocytes by western blot and immunofluorescence. This study contributes to enlarge the existing knowledge related to NOS structure and NOS participation in the immune response in lobsters. The evaluation of an antibody capable to recognize NOS from lobsters constitutes a novel and interesting tool for the implementation of further studies on NOS functions in crustaceans.