A natural prodrug activation mechanism in nonribosomal peptide synthesis

We have identified a new mechanism for the cleavage and activation of nonribosomally made peptides and peptide-polyketide hybrids that are apparently operational in several different bacteria. This process includes the cleavage of a precursor molecule by a membrane-bound and D-asparagine-specific peptidase, as shown here in the biosynthesis of the antibiotic xenocoumacin from Xenorhabdus nematophila.     

Mutational analysis of the C-domain in nonribosomal peptide synthesis.

The initial condensation event in the nonribosomal biosynthesis of the peptide antibiotics gramicidin S and tyrocidine A takes place between a phenylalanine activating racemase GrsA/TycA and the first proline-activating module of GrsB/TycB. Recently we established a minimal in vitro model system for NRPS with recombinant His6-tagged GrsA (GrsAPhe-ATE; 127 kDa) and TycB1 (TycB1Pro-CAT; 120 kDa) and demonstrated the catalytic function of the C-domain in TycB1Pro-CAT to form a peptide bond between phenylalanine and proline during diketopiperazine formation (DKP). In this work we took advantage of this system to identify catalytically important residues in the C-domain of TycB1Pro-CAT using site-directed mutagenesis and peptide mapping. Mutations in TycB1Pro-CAT of 10 strictly conserved residues among 80 other C-domains with potential catalytic function, revealed that only R62A, H147R and D151N are impaired in peptide-bond formation. All other mutations led to either unaffected (Q19A, C154A/S, Y166F/W and R284A) or insoluble proteins (H146A, R67A and W202L). Although 100 nm of the serine protease inhibitors N-alpha-tosyl-l-phenylalanylchloromethane or phenylmethanesulfonyl fluoride completely abolished DKP synthesis, no covalently bound inhibitor derivatives in the C-domain could be identified by peptide mapping using HPLC-MS. Though the results do not reveal a particular mechanism for the C-domain, they exhibit a possible way of catalysis analogous to the functionally related enzymes chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. Based on this, we propose a mechanism in which one catalytic residue (H147) and two other structural residues (R62 and D151) are involved in amino-acid condensation.     

Amino acid activation and polymerization at modular multienzymes in nonribosomal peptide biosynthesis

Summary The biosynthesis of microbial bioactive peptides is accomplished nonribosomally by large multifunctional enzymes consisting of linearly arranged building blocks of 1,000–1,500 amino acid residues. Each of these units acts as an independent enzyme which catalyzes the selection, activation, and in some cases modification (epimerization, N-methylation) of its cognate amino acid, as well as the elongation of the peptide product. The specific linkage of amino acid activating modules upon such polyenzymes defines the sequence of the peptide product. A series of functional domains could be identified upon an amino acid activating module which are involved in the sequential reactions in nonribosomal peptide biosynthesis.Abbrevations aaRS aminoacyl tRNA synthetase - GS1 gramicidin S synthetase 1 (phenylalanine racemase) - GS2 gramicidin S synthetase 2 - TY1 and 2 tyrocidine synthetase 1 and 2 - ACV [-(l--aminoadipyl)-l-cysteinyl-d-valine] - FITC fluorescein 5-isothiocyanate - FAB-, ESI-MS fast atom bombardment-, electrospray ionization-mass spectrometry - Pan 4-phosphopantetheine - NMR nuclear magnetic resonance - ACP acyl carrier protein - SAM S-adenosyl-l-methionine - CM carboxy-methyl - NES Nethylsuccinimido     

Dissecting and exploiting nonribosomal peptide synthetases

Over the past decade striking advances in microbialgenetics have propelled a revolution in our ability todeduce, analyze and manipulate the biosynthesis of struc-turally complex and biologically important families of na-ture products, one most notable cla…     

Relationship between fatty acid binding proteins,acetyl-CoA formation and fatty acid synthesis in developing human placenta

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-¹⁴ C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.     

Functional cross-talk between two-component and phytochrome B signal transduction in Arabidopsis

The A-type response regulator ARR4 is an element in the two-component signalling network of Arabidopsis. ARR4 interacts with the N-terminus of the red/far-red light photoreceptor phytochrome B (phyB) and functions as a modulator of photomorphogenesis. In concert with other A-type response regulators, ARR4 also participates in the modulation of the cytokinin response pathway. Here evidence is presented that ARR4 directly modulates the activity state of phyB in planta, not only under inductive but also under extended irradiation with red light. Mutation of the phosphorylatable aspartate to asparagine within the receiver domain creates a version of ARR4 that negatively affects photomorphogenesis. Additional evidence suggests that ARR4 activity is regulated by a phosphorelay mechanism that depends on the AHK family of cytokinin receptors. Accordingly, the ability of ARR4 to function on phyB is modified by exogenous application of cytokinin. These results implicate a cross-talk between cytokinin and light signalling mediated by ARR4. This cross-talk enables the plant to adjust light reponsiveness to endogenous requirements in growth and development.     

Interrelationships between fatty acid biosynthesis and acyl-lipid synthesis in Chlorella vulgaris

1. Fatty acid synthesis from [2-(14)C]acetate by Chlorella vulgaris cells grown and incubated in the dark is limited almost entirely to the production of saturated and monoenoic acids. 2. In light-incubated cells, both saturated and polyunsaturated fatty acids are rapidly synthesized. 3. Two groups of lipids can be distinguished in both dark- and light-incubated cells. The first group, consisting of phosphatidyl-glycerol, monogalactosyl diglyceride, lecithin and neutral glyceride, has a very high turnover rate for certain fatty acids. The second group, consisting of digalactosyl diglyceride, sulpholipid, phosphatidylethanolamine and phosphatidylinositol, has a slow turnover of fatty acids. 4. The lipids with rapid fatty acid turnover may be involved in the sequences of saturated and unsaturated fatty acid synthesis. A classification of lipids is made on the basis of their suggested functions.     

Fatty acid synthesis by spinach chloroplasts III. Relationship between fatty acid synthesis and photophosphorylation

The modes of actions of photosynthetic inhibitors on photosynthesisand fatty acid synthesis were examined. DCMU, an electron transport inhibitor, inhibited fatty acidsynthesis and photophosphorylation to the same extent, suggestingdependence of fatty acid synthesis on photosynthesis. The samewas also the case with FCCP, a photophosphorylation uncoupler.In contrast, NH₄Cl and phlorizin at concentrations completelysuppressing ATP formation, only partially inhibited the fattyacid synthesis. These facts suggest that a certain level ofhigh-energy intermediate (state) is responsible for the lightenhancement of fatty acid synthesis. This idea is further supportedby the fact that the partial inhibition of fatty acid synthesisby NH₄Cl was relieved by addition of DCCD at low concentrationssuppressing the ATP formation but not completely destroyingthe high energy intermediate. The lag period in the initial period of fatty acid synthesiswas shortened by preillumination of chloroplasts, even in theabsence of ADP. This indicates that the light dependent fattyacid synthesis is closely associated with the high-energy intermediate(state), but not directly with ATP formation by photophosphorylation. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis

Nonribosomal peptide synthetases (NRPSs) make many natural products of clinical importance, but a deeper understanding of the protein domains that compose NRPS assembly lines is required before these megasynthetases can be effectively engineered to produce novel drugs. The N-terminal amide bond-forming condensation (C) domain of the enterobactin NRPS EntF was excised from the multidomain synthetase using endpoints determined from sequence alignments and secondary structure predictions. The isolated domain was well-folded when compared by circular dichroism to the vibriobactin NRPS VibH, a naturally free-standing C domain. The EntF domain was also fully functional in an assay based on a synthetic small-molecule substrate, seryl N-acetylcysteamine. Active site mutants of the EntF C domain were surprisingly inactive in vitro as compared to their VibH counterparts, yet maintained the overall domain structure. An in vivo assay was developed in the context of the full-length EntF protein to more sensitively probe the activity level of the C domain mutants, and this supported strong effects for the active site mutations. The crucial role of histidine-138 was confirmed by assay of the full-length protein in vitro. These results suggest a strong resemblance of catalysis by the EntF C domain to chloramphenicol acetyltransferase, including an active site organized by an arginine-aspartate salt bridge, a key histidine acting as a general base, and an asparagine instead of a serine stabilizing the proposed tetrahedral intermediate by hydrogen bonding. The precise definition of a functional C domain excised from a NRPS should aid efforts at swapping NRPS domains between assembly lines.     

Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis.

Recent studies on type II thioesterases (TEIIs) involved in microbial secondary metabolism described a role for these enzymes in the removal of short acyl-S- phosphopantetheine intermediates from misprimed holo-(acyl carrier proteins) and holo-(peptidyl carrier proteins) of polyketide synthases and nonribosomal peptide synthetases. Because of the absence of structural information on this class of enzymes, we performed a mutational analysis on a prototype TEII essential for efficient production of the lipopeptide antibiotic surfactin (TEII(srf)), which led to identification of catalytic and structural residues. On the basis of sequence alignment of 16 TEIIs, 10 single and one double mutant of highly conserved residues of TEII(srf) were constructed and biochemically investigated. We clearly identified a catalytic triad consisting of Ser86, Asp190 and His216, suggesting that TEII(srf) belongs to the alpha/beta-hydrolase superfamily. Exchange of these residues with residues with aliphatic side chains abolished enzyme activity, whereas replacement of the active-site Ser86 with cysteine produced an enzyme with marginally reduced activity. In contrast, exchange of the second strictly conserved asparagine (Asp163) with Ala resulted in an active but unstable enzyme, excluding a role for this residue in catalysis and suggesting a structural function. The results define three catalytic and at least one structural residue in a nonribosomal peptide synthetase TEII.     

Evidence for cross-talk between atrial natriuretic peptide and nitric oxide receptors

Guanylyl cyclases (GCs), a ubiquitous family of enzymes that metabolize GTP to cyclic GMP (cGMP), are traditionally divided into membrane-bound forms (GC-A-G) that are activated by peptides and cytosolic forms that are activated by nitric oxide (NO) and carbon monoxide. However, recent data has shown that NO activated GC’s (NOGC) also may be associated with membranes. In the present study, interactions of guanylyl cyclase A (GC-A), a caveolae-associated, membrane-bound, homodimer activated by atrial natriuretic peptide (ANP), with NOGC, a heme-containing heterodimer (α/β) β1 isoform of the β subunit of NOGC (NOGCβ1) was specifically focused. NOGCβ1 co-localized with GC-A and caveolin on the membrane in human kidney (HK-2) cells. Interaction of GC-A with NOGCβ1 was found using immunoprecipitations. In a second set of experiments, the possibility that NOGCβ1 regulates signaling by GC-A in HK-2 cells was explored. ANP-stimulated membrane guanylyl cyclase activity (0.05 ± 0.006 pmol/mg protein/5 min; P < 0.01) and intra cellular GMP (18.1 ± 3.4 vs. 1.2 ± 0.5 pmol/mg protein; P < 0.01) were reduced in cells in which NOGCβ1 abundance was reduced using specific siRNA to NOGCβ1. On the other hand, ANP-stimulated cGMP formation was increased in cells transiently transfected with NOGCβ1 (530.2 ± 141.4 vs. 26.1 ± 13.6 pmol/mg protein; P < 0.01). siRNA to NOGCβ1 attenuated inhibition of basolateral Na/K ATPase activity by ANP (192 ± 22 vs. 92 ± 9 nmol phosphate/mg protein/min; P < 0.05). In summary, the results show that NOGCβ1 and GC-A interact and that NOGCβ1 regulates ANP signaling in HK-2 cells. The results raise the novel possibility of cross-talk between NOGC and GC-A signaling pathways in membrane caveolae.     

Natural roles of nonribosomal peptide metabolites in fungi

Over 90 years have passed since Alexander Fleming's discovery of penicillin, the first recognized, naturally occurring antibiotic. Penicillin is a representative of a group of metabolites produced by large multienzyme complexes [nonribosomal peptide synthetases (NRPSs)] in a ribosome-independent fashion. Nonribosomal peptides (NRPs) are structurally diverse metabolites produced almost exclusively by bacteria and fungi. NRPs include bioactive compounds useful for pharmaceutical applications (e.g., antibiotics, antitumor compounds, and immunosuppressants) and therefore much progress has been made in our understanding of medically relevant characteristics of NRPs in the past decades. Natural roles of NRP metabolites, on the other hand, have been largely ignored, and much less is known about the biological/physiological significance of NRPs under natural settings. In the present review, we summarize past and current work on natural functions of NRPs in their fungal producers, with a focus on virulence, development, and stress tolerance, and highlight the diverse roles these small peptide metabolites play. Some NRPs are involved in interactions with host organisms, others work in fungus-environment interfaces, and still others are crucial for vegetative and reproductive development of the producing fungi.     

Relationship between carbohydrate-induced hypertriglyceridemia and fatty acid synthesis in lean and obese subjects

We previously reported that a eucaloric, low fat, liquid formula diet enriched in simple carbohydrate markedly increased the synthesis of fatty acids in lean volunteers. To examine the diet sensitivity of obese subjects, 7 obese and 12 lean volunteers were given two eucaloric low fat solid food diets enriched in simple sugars for 2 weeks each in a random-order, cross-over design (10% fat, 75% carbohydrate vs. 30% fat, 55% carbohydrate, ratio of sugar to starch 60:40). The fatty acid compositions of both diets were matched to the composition of each subject's adipose tissue and fatty acid synthesis measured by the method of linoleate dilution in plasma VLDL triglyceride. In all subjects, the maximum % de novo synthesized fatty acids in VLDL triglyceride 3;-9 h after the last meal was higher on the 10% versus the 30% fat diet. There was no significant difference between the dietary effects on lean (43+/-13 vs. 12+/-13%) and obese (37+/-15 vs. 6+/-6%) subjects, despite 2-fold elevated levels of insulin and reduced glucagon levels in the obese. Similar results were obtained for de novo palmitate synthesis in VLDL triglyceride measured by mass isotopomer distribution analysis after infusion of [(13)C]acetate. On the 10% fat diet, plasma triglycerides (fasting and 24 h) were increased and correlated with fatty acid synthesis. Triglycerides were higher when fatty acid synthesis was constantly elevated rather than having diurnal variation.Thus, eucaloric, solid food diets which are very low in fat and high in simple sugars markedly stimulate fatty acid synthesis from carbohydrate, and plasma triglycerides increase in proportion to the amount of fatty acid synthesis. However, this dietary effect is not related to body mass index, insulin, or glucagon levels.     

水杨酸、茉莉酸和乙烯介导的防卫信号途径相互作用的研究进展

乙烯、水杨酸和茉莉酸是植物体内主要的几个防御信号途径,也是研究比较多的几个信号途径。很多试验证明不同的防御信号途径相互间存在相互作用,他们或相互抑制,或相互促进。从这三种信号途径相互间的作用,及作用的联系点进行综述。     

Proteomic analysis of polyketide and nonribosomal peptide biosynthesis

Polyketides and non-ribosomal peptides are in a class of natural products important both as drug sources and as dangerous toxins and virulence factors. While studies over the last two decades have provided substantial characterization of the modular synthases that produce these compounds at the genetic level, their understanding at the protein level is much less understood. New proteomic platforms called an orthogonal active site identification system (OASIS) and proteomic interrogation of secondary metabolism (PrISM) have been developed to identify and quantify natural product synthase enzymes. Reviewed here, these tools offer the means to discover and analyze modular synthetic pathways that are limited by genetic techniques, opening the tools of contemporary proteomics to natural product sciences.     

Carrier protein recognition in siderophore-producing nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) use phosphopantetheine (pPant) bearing carrier proteins to chaperone activated aminoacyl and peptidyl intermediates to the various enzymes that effect peptide synthesis. Using components from siderophore NRPSs that synthesize vibriobactin, enterobactin, yersiniabactin, pyochelin, and anguibactin, we examined the nature of the interaction of such cofactor-carrier proteins with acyl-activating adenylation (A) domains. While VibE, EntE, and PchD were all able to utilize "carrier protein-free" pPant derivatives, the pattern of usage indicated diversity in the binding mechanism, and even the best substrates were down at least 3 log units relative to the native cofactor-carrier protein. When tested with four noncognate carrier proteins, EntE and VibE differed both in the range of substrate utilization efficiency and in the distribution of the efficiencies across this range. Correlating sequence alignments to kinetic efficiency allowed for the construction of eight point mutants of VibE's worst substrate, HMWP2 ArCP, to the corresponding residue in its native VibB. Mutants S49D and H66E combined to increase activity 6.2-fold and had similar enhancing effects on the downstream condensation domain VibH, indicating that the two NRPS enzymes share carrier protein recognition determinants. Similar mutations of HMWP2 ArCP toward EntB had little effect on EntE, suggesting that the position of recognition determinants varies across NRPS systems.     

Mitochondrial fatty acid synthesis and respiration

Recent studies have revealed that mitochondria are able to synthesize fatty acids in a malonyl-CoA/acyl carrier protein (ACP)-dependent manner. This pathway resembles bacterial fatty acid synthesis (FAS) type II, which uses discrete, nuclearly encoded proteins. Experimental evidence, obtained mainly through using yeast as a model system, indicates that this pathway is essential for mitochondrial respiratory function. Curiously, the deficiency in mitochondrial FAS cannot be complemented by inclusion of fatty acids in the culture medium or by products of the cytosolic FAS complex. Defects in mitochondrial FAS in yeast result in the inability to grow on nonfermentable carbon sources, the loss of mitochondrial cytochromes a/a3 and b, mitochondrial RNA processing defects, and loss of cellular lipoic acid. Eukaryotic FAS II generates octanoyl-ACP, a substrate for mitochondrial lipoic acid synthase. Endogenous lipoic acid synthesis challenges the hypothesis that lipoic acid can be provided as an exogenously supplied vitamin. Purified eukaryotic FAS II enzymes are catalytically active in vitro using substrates with an acyl chain length of up to 16 carbon atoms. However, with the exception of 3-hydroxymyristoyl-ACP, a component of respiratory complex I in higher eukaryotes, the fate of long-chain fatty acids synthesized by the mitochondrial FAS pathway remains an enigma. The linkage of FAS II genes to published animal models for human disease supports the hypothesis that mitochondrial FAS dysfunction leads to the development of disorders in mammals.     

Mechanism of fatty acid synthesis

Significant advances have been made in the past few years in our understanding of the mechanism of synthesis of fatty acids, the structural organization of fatty acid synthetase complexes and the mechanism of regulation of activity of these enzyme systems. Numerous fatty acid synthetase complexes have been purified to homogeneity and the mechanism of synthesis of fatty acids by these enzyme systems has been ascertained from tracer, and recently, kinetic studies. The results obtained by these methods are in complete agreement. Furthermore, the kinetic results have indicated that fatty acid synthesis proceeds by a seven-site ping-pong mechanism. Several of the fatty acid synthetases have been dissociated completely to nonidentical half-molecular weight subunit species and these have been separated by affinity chromatography. From one of these subunits acyl carrier protein has been obtained. Whether the nonidentical subunits can be dissociated into individual proteins or whether these subunits are each comprised of one peptide is still a matter of controversy. However, it appears to us that each of the half-molecular weight subunits is indeed comprised of individual proteins. Studies on the regulation of activity of fatty acid synthetase complexes of avian and mammalian liver have resulted in the separation by affinity chromatography of three species (apo, holo-$\text{a}$ and holo-$\text{b}$) of fatty acid synthetase. Since these species have radically different enzyme activities they may provide a mechanism of short-term regulation of fatty acid synthetase activity. Other studies have shown that the quantity of avian and mammalian liver fatty acid synthetases is controlled by a change in the rate of synthesis of this enzyme complex. This change in the rate of synthesis of enzyme complex is under the control of insulin and glucagon. The former hormone increases the rate of enzyme synthesis, whereas the latter decreases it. Further studies on fatty acid synthetase complexes will undoubtedly concentrate upon more refined aspects of the structural organization of these enzyme systems, including the sequencing of acyl carrier proteins, the effects of protein-protein interaction on the kinetics of the partial reactions of fatty acid synthesis catalyzed by separated enzymes of the complex, the mechanism of hormonal regulation of fatty acid synthetase activity and x-ray diffraction analysis of subunits and complex.