Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines.

Nonribosomal peptide synthetases are large enzyme complexes that synthesize a variety of peptide natural products through a thiotemplated mechanism. Assembly of the peptides proceeds through amino acid loading, amide-bond formation and chain translocation, and finally thioester lysis to release the product. The final products are often heavily modified, however, through methylation, epimerization, hydroxylation, heterocyclization, oxidative cross-linking and attachment of sugars. These activities are the province of specialized enzymes (either embedded in the multidomain nonribosomal peptide synthetase structure or standalone).

Heterocycle formation in vibriobactin biosynthesis: alternative substrate utilization and identification of a condensed intermediate.

The iron-chelating peptide vibriobactin of the pathogenic Vibrio cholerae is assembled by a four-subunit nonribosomal peptide synthetase complex, VibE, VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as precursors to two 2,3-dihydroxyphenyl- (DHP-) methyloxazolinyl groups in amide linkage on a norspermidine scaffold. We have tested the ability of the six-domain VibF subunit (Cy-Cy-A-C-PCP-C) to utilize various L-threonine analogues and found the beta-functionalized amino acids serine and cysteine can function as alternate substrates in aminoacyl-AMP formation (adenylation or A domain), aminoacyl-S-enzyme formation (A domain), acylation by 2,3-dihydrobenzoyl- (DHB-) S-VibB (heterocyclization or Cy domain), heterocyclization to DHP-oxazolinyl- and DHP-thiazolinyl-S-enzyme forms of VibF (Cy domain) as well as transfer to DHB-norspermidine at both N(5) and N(9) positions (condensation or C domain) to make the bis(oxazolinyl) and bis(thiazolinyl) analogues of vibriobactin. When L-threonyl-S-pantetheine or L-threonyl-S-(N-acetyl)cysteamine was used as a small-molecule thioester analogue of the threonyl-S-VibF acyl enzyme intermediate, the Cy domain(s) of a CyCyA fragment of VibF generated DHB-threonyl-thioester products of the condensation step but not the methyloxazolinyl thioesters of the heterocyclization step. This clean separation of condensation from cyclization validates a two-stage mechanism for threonyl, seryl, and cysteinyl heterocyclization domains in siderophore and antibiotic synthetases. Full heterocyclization activity could be restored by providing CyCyA with the substrate L-threonyl-S-peptidyl carrier protein (PCP)-C2, suggesting an important role for the protein scaffold component of the heterocyclization acceptor substrate. We also examined heterocyclization donor substrate specificity at the level of acyl group and protein scaffold and observed intolerance for substitution at either position.

Reconstitution and characterization of the Vibrio cholerae vibriobactin synthetase from VibB, VibE, VibF, and VibH.

Vibriobactin [N(1)-(2,3-dihydroxybenzoyl)-N(5),N(9)-bis[2-(2, 3-dihydroxyphenyl)-5-methyloxazolinyl-4-carboxamido]norspermidine] , is an iron chelator from the cholera-causing bacterium Vibrio cholerae. The six-domain, 270 kDa nonribosomal peptide synthetase (NRPS) VibF, a component of vibriobactin synthetase, has been heterologously expressed in Escherichia coli and purified. VibF has an unusual NRPS domain organization: cyclization-cyclization-adenylation-condensation-peptidyl carrier protein-condensation (Cy(1)-Cy(2)-A-C(1)-PCP-C(2)). VibF activates and covalently loads its PCP with L-threonine, and together with vibriobactin synthetase proteins VibE (adenylation) and VibB (aryl carrier protein) condenses and heterocyclizes 2, 3-dihydroxybenzoyl-VibB with L-Thr to 2-dihydroxyphenyl-5-methyloxazolinyl-4-carboxy-VibF in the first demonstration of oxazoline formation by an NRPS cyclization domain. This enzyme-bound aryl oxazoline can be transferred by VibF to various amine acceptors but most efficiently to N(1)-(2, 3-dihydroxybenzoyl)norspermidine (k(cat) = 122 min(-1), K(m) = 1.7 microM), the product of 2,3-dihydroxybenzoyl-VibB, norspermidine, and VibH. This diacylated product undergoes a second aryl oxazoline acylation on its remaining secondary amine, also catalyzed by VibF, to yield vibriobactin. Vibriobactin biosynthesis in vitro has thus been accomplished from four proteins, VibE, VibB, VibF, and VibH, with the substrates 2,3-dihydroxybenzoic acid, L-Thr, norspermidine, and ATP. Vibriobactin synthetase is an unusual NRPS in that all intermediates are not covalently tethered as PCP thioesters and in that it represents an NRPS pathway with two branch points.

Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains.

The Vibrio cholerae siderophore vibriobactin is biosynthesized from three molecules of 2,3-dihydroxybenzoate (DHB), two molecules of L-threonine, and one of norspermidine. Of the four genes positively implicated in vibriobactin biosynthesis, we have here expressed, purified, and assayed the products of three: vibE, vibB, and vibH. All three are homologous to nonribosomal peptide synthetase (NRPS) domains: VibE is a 2,3-dihydroxybenzoate-adenosyl monophosphate ligase, VibB is a bifunctional isochorismate lyase-aryl carrier protein (ArCP), and VibH is a novel amide synthase that represents a free-standing condensation (C) domain. VibE and VibB are homologous to EntE and EntB from Escherichia coli enterobactin synthetase; VibE activates DHB as the acyl adenylate and then transfers it to the free thiol of the phosphopantetheine arm of VibB's ArCP domain. VibH then condenses this DHB thioester (the donor) with the small molecule norspermidine (the acceptor), forming N(1)-(2, 3-dihydroxybenzoyl)norspermidine (DHB-NSPD) with a k(cat) of 600 min(-1) and a K(m) for acyl-VibB of 0.88 microM and for norspermidine of 1.5 mM. Exclusive monoacylation of a primary amine of norspermidine was observed. VibH also tolerates DHB-acylated EntB and 1,7-diaminoheptane, octylamine, and hexylamine as substrates, albeit at lowered catalytic efficiencies. DHB-NSPD possesses one of three acylations required for mature vibriobactin, and its formation confirms VibH's role in vibriobactin biosynthesis. VibH is a unique NRPS condensation domain that acts upon an upstream carrier-protein-bound donor and a downstream amine, turning over a soluble amide product, in contrast to an archetypal NRPS-embedded C domain that condenses two carrier protein thioesters.

Molecular mechanism of VanHst, an alpha-ketoacid dehydrogenase required for glycopeptide antibiotic resistance from a glycopeptide producing organism.

The vancomycin resistance enzyme VanH is an alpha-ketoacid dehydrogenase that stereospecifically reduces pyruvate to D-lactate, which is required for the synthesis of the depsipeptide D-alanine-D-lactate. This compound then forms an integral part of the bacterial cell wall replacing the vancomycin target dipeptide D-alanine-D-alanine, thus the presence of VanH is essential for glycopeptide resistance. In this work, the VanH homologue from the glycopeptide antibiotic producing organism Streptomyces toyocaensis NRRL 15009, VanHst, has been overexpressed in Escherichia coli and purified, and its substrate specificity and mechanism were probed by steady-state kinetic methods and site-directed mutagenesis. The enzyme is highly efficient at pyruvate reduction with kcat/Km = 1.3 x 10(5) M-1 s-1 and has a more restricted alpha-ketoacid substrate specificity than VanH from vancomycin resistant enterococci (VRE). Conversely, VanHst shows no preference between NADH and NADPH while VanH from VRE prefers NADPH. The kinetic mechanism for VanHst was determined using product and dead-end inhibitors to be ordered BiBi with NADH binding first followed by pyruvate and products leaving in the order D-lactate, NAD+. Site-directed mutagenesis indicated that Arg237 plays a role in pyruvate binding and catalysis and that His298 is a candidate for an active-site proton donor. Glu266, which has been suggested to modulate the pKa of the catalytic His in other D-lactate dehydrogenases, was found to fulfill a similar role in VanHst, lowering a pKa value of kcat/Km nearly 2 units. These results now provide the framework for additional structure and inhibitor design work on the VanH family of antibiotic resistance enzymes.

DdlN from vancomycin-producing Amycolatopsis orientalis C329.2 is a VanA homologue with D-alanyl-D-lactate ligase activity.

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in D-alanyl-D-lactate. A key enzyme in this process is a D-alanyl-D-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide D-alanyl-D-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for D-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.

Glycopeptide antibiotic resistance genes in glycopeptide-producing organisms.

The mechanism of high-level resistance to vancomycin in enterococci consists of the synthesis of peptidoglycan terminating in D-alanyl-D-lactate instead of the usual D-alanyl-D-alanine. This alternate cell wall biosynthesis pathway is ensured by the collective actions of three enzymes: VanH, VanA, and VanX. The origin of this resistance mechanism is unknown. We have cloned three genes encoding homologs of VanH, VanA, and VanX from two organisms which produce glycopeptide antibiotics: the A47934 producer Streptomyces toyocaensis NRRL 15009 and the vancomycin producer Amycolatopsis orientalis C329.2. The predicted amino acid sequences are highly similar to those found in VRE: 54 to 61% identity for VanH, 59 to 63% identity for VanA, and 61 to 64% identity for VanX. Furthermore, the orientations of the genes, vanH, vanA, and vanX, are identical to the orientations found in vancomycin-resistant enterococci. Southern analysis of total DNA from other glycopeptide-producing organisms, A. orientalis 18098 (chloro-eremomycin producer), A. orientalis subsp. lurida (ristocetin producer), and Amycolatopsis coloradensis subsp. labeda (teicoplanin and avoparcin producer), with a probe derived from the vanH, vanA, and vanX cluster from A. orientalis C329.2 revealed cross-hybridizing DNA in all strains. In addition, the vanH, vanA, vanX cluster was amplified from all glycopeptide-producing organisms by PCR with degenerate primers complementary to conserved regions in VanH and VanX. Thus, this gene sequence is common to all glycopeptide producers tested. These results suggest that glycopeptide-producing organisms may have been the source of resistance genes in vancomycin-resistant enterococci.

D-Ala-D-Ala ligases from glycopeptide antibiotic-producing organisms are highly homologous to the enterococcal vancomycin-resistance ligases VanA and VanB.

The crisis in antibiotic resistance has resulted in an increasing fear of the emergence of untreatable organisms. Resistance to the glycopeptide antibiotic vancomycin in the enterococci, and the spread of these pathogens throughout the environment, has shown that this scenario is a matter of fact rather than fiction. The basis for vancomycin resistance is the manufacture of the depsipeptide D-Ala-D-lactate, which is incorporated into the peptidoglycan cell wall in place of the vancomycin target D-Ala-D-Ala. Pivotal to the resistance mechanism is the production of a D-Ala-D-Ala ligase capable of ester formation. Two highly efficient depsipeptide ligases have been cloned from vancomycin-resistant enterococci: VanA and VanB. These ligases show high amino acid sequence similarity to each other ( approximately 75%), but less so to other D-Ala-D-X ligases (<30%). We have cloned ddls from two glycopeptide-producing organisms, the vancomycin producer Amycolatopsis orientalis and the A47934 producer Streptomyces toyocaensis. These ligases show strong predicted amino acid homology to VanA and VanB (>60%) but not to other D-Ala-D-X ligases (<35%). The D-Ala-D-Ala ligase from S. toyocaensis shows D-Ala-D-lactate synthase activity in cell-free extracts of S. lividans transformed with the ddl gene and confirms the predicted enzymatic activity. These results imply a close evolutionary relationship between resistance mechanisms in the clinics and in drug-producing bacteria.

The glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009 has both D-alanyl-D-alanine and D-alanyl-D-lactate ligases.

High level resistance to vancomycin and other glycopeptide antibiotics requires the synthesis of peptidoglycan terminating in the depsipeptide D-Ala-D-lactate, rather the usual D-Ala-D-Ala. We report the purification and enzymatic characterization of two D-Ala ligases from Streptomyces toyocaensis NRRL 15009 which produces the glycopeptide antibiotic A47934. One of these enzymes catalyzes only D-Ala-D-Ala peptide formation and is recovered from mid-exponential phase cell cultures. The other enzyme is a D-Ala-D-lactate ligase which can be detected in actively antibiotic producing stationary phase cultures or mid-exponential phase cultures grown in the presence of A47934. These results imply that peptidoglycan components of S. toyocaensis NRRL 15009 change upon induction of antibiotic production and predict the existence of a VanX-like D-Ala-D-Ala DD-dipeptidase activity.

Activation of ion channels in the frog endplate by several analogues of acetylcholine.

1. Single-ion-channel recording has been used to estimate the equilibrium concentration-response relationship for several acetylcholine analogues. The response, corrected for desensitization, was taken as the probability of a channel being open during clusters of openings that were separated by desensitized periods. 2. All agonists were able to block the channels which they themselves opened. Carbachol, suberyldicholine and the sulphonium analogue of acetylcholine were all found to be efficacious agonists in the sense that the results indicate that all of them, in sufficiently high concentration, would be able to open 90% or more of channels if it were not for channel block. 3. In the case of suberyldicholine the results are much as predicted by the interpretation of the fine structure of channel openings at low agonist concentrations. 4. The maximum probability of opening that could be obtained with decamethonium and with phenyltrimethylammonium was low (below 4%), and it was not possible to distinguish whether this was wholly a result of the powerful (relative to activation potency) channel-blocking action of these agonists, or whether it was to some extent attributable to their being genuine partial agonists. 5. The results suggest that, for a range of agonists, differences in equilibrium potency are usually more strongly influenced by affinity for binding to the resting state of the receptor than by ability to activate the receptor once bound, though in the case of suxamethonium (relative to acetylcholine) the contributions of each factor are similar.

The actions of suxamethonium (succinyldicholine) as an agonist and channel blocker at the nicotinic receptor of frog muscle.

1. Patch clamp methods were used to study the equilibrium and kinetic properties of the acetylcholine analogue, succinyldicholine (suxamethonium), which is used clinically as a neuromuscular blocking agent. 2. The equilibrium concentration-response curve, corrected for desensitization was estimated by measuring as the response the probability of being open of single ion channels during clusters of activity that occur between long desensitized periods. Suxamethonium (Sux) was about 7.6-fold less potent than acetylcholine (ACh) (at low concentrations), partly because of 2.9-fold lower affinity for the resting receptor, and partly because of a lower ability to activate the receptor once bound. 3. Sux was a more potent blocker of the open ion channel than ACh (equilibrium constant about 200 microM); this limited the maximum open probability to about 0.36 (at 12 degrees C and -120 mV). Individual channel blockages lasted about 65 microseconds on average. They appeared to get longer at high agonist concentration; however, a simulation method was used to show that this effect could be accounted for by the fact that at higher concentrations there are more openings that are too brief to be detected. Over the concentration range tested the effects were described by a simple open channel block mechanism. 4. No component of brief shut times could be detected other than those resulting from channel blockages. However, the results suggest that multiple channel openings (the nachschlag phenomenon) should be rare, so this is not inconsistent with previous results with other agonists. 5. Sux differed from ACh and carbachol in that it had a somewhat lower efficacy and a greater channel blocking action. However, in clinical practice channel block is unlikely to contribute to neuromuscular block to any significant extent; the main mechanism of paralysis, at least in the early stages, is probably a result of prolonged depolarization of the region of membrane surrounding the motor endplate leading to inactivation of the sodium channels therein.

Voltage-activated membrane currents in rat cerebellar granule neurones.

1. Voltage-activated currents have been recorded from cerebellar granule neurones in explant cultures from young rats (1-9 days old). Cells were examined with whole-cell patch-clamp methods. Depolarizing pulses from a pre-pulse potential of -100 mV evoked a rapidly activated transient inward current, and an outward current which decayed in two phases. The ionic dependence, kinetics and pharmacological properties of these currents have been studied. 2. Peak inward Na+ currents in cells from 7-day-old rats were in the range 350-450 pA. No evidence was found for the presence of calcium currents. Thus, inward current was unchanged in zero Ca2+, 1 mM-EGTA solution. No inward current was obtained in medium containing 10 mM-Ba2+ and tetrodotoxin (TTX). Supplementing the pipette (i.e. intracellular) solution with Mg-ATP did not reveal any Ca2+ current. 3. Depolarizing steps (from -100 mV) in TTX-containing solution gave an early transient outward current and a late outward current. The transient current resembled IA described in other cells, and reversed close to EK in both normal and elevated potassium concentrations, indicating that K+ is the predominant charge carrier. Depolarizing steps from -50 mV failed to give a transient outward current, and gave only a slowly rising current which resembled the late potassium current, IK. 4. Inactivation of the transient current was examined by applying test depolarizations from increasingly negative pre-pulse potentials (-50 to -120 mV): half-inactivation occurred at -72 mV. Transient outward currents decayed exponentially with time constants, tau, of 7.3-25.3 ms at 0 mV. The time course of removal of inactivation in cells held at -50 mV, and given increasingly long pre-pulses to -100 mV, was exponential with tau = 35 ms. 5. Both transient and late outward currents were reversibly abolished by addition to the bathing medium of 10 mM-Ba2+ or 1 mM-quinine. Outward K+ current was not dependent on external calcium. Tetraethylammonium (20 mM) selectively reduced the late outward current; the peak transient current was reduced by less than 20%. 4-Aminopyridine (2 mM) showed little selectivity between transient and late outward currents. 6. It is concluded that cerebellar granule cells from young rats possess voltage-activated inward Na+ current as well as two types of K+ current, IA and IK. In terms of neuronal functioning, the properties of the transient outward current may confer a role in regulating excitability and in repolarization, but a definitive statement will require knowledge of the cellular location and relative densities of channels in granule cells in vivo.

Excitability and secretory activity in the salivary gland cells of jawed leeches (Hirudinea: Gnathobdellida).

Thousands of salivary cells fill the interstices throughout the anterior ends of jawed leeches. The somata are large (30-200 micron in diameter). They project single processes (ductules) into the three jaws, and were found to fire overshooting action potentials of 50-85 mV amplitude and 100-200 ms duration at low spontaneous rates. The action potentials were not detected in the presence of cobalt (10 mmol l-1), but could be recorded when sodium was absent from the Ringer, so they appear to be calcium-dependent. Salivary material is transported by the long processes of these unicellular glands and secreted into ducts which alternate with paired teeth on the jaws. Secretion is activated reliably by 10(-6) mol l-1 serotonin, but not by other neurotransmitters found in the leech nervous system. Each jaw secretes at an average rate of 230 nl min-1 in the presence of serotonin, and secretion is completely abolished by cobalt. Perfusion with serotonin excites the salivary gland cells into impulse activity, and often evokes bursting. Impulse activity of the peripherally projecting, serotonergic Retzius cells evokes both depolarizations and action potentials in the salivary gland cells. In jawed leeches, central neurones appear to control salivation by a peripheral release of serotonin. This neurotransmitter evokes calcium-dependent action potentials and calcium, in turn, stimulates secretion.

Calcium-dependent action potentials in leech giant salivary cells.

Two pairs of discrete salivary glands are located at the base of the muscular proboscis of the sanguivorous Glossiphoniid leeches Haementeria ghilianii and Haementeria officinalis. Each anterior gland is 0.8 cm to 2 cm in length, and comprises over 200 giant salivary cell bodies ranging from 150 microns to over 1000 microns in diameter, depending on the size of the animal. The salivary cells are neither electrically nor dye coupled, and there is no acinar structure or common duct, but instead each cell extends an individual ductule. The cells fire action potentials of 100-200 ms duration and 70-100 mV amplitude in response to depolarizing pulses, or at the cessation of a hyperpolarizing pulse. The impulse is abolished by procedures known to antagonize calcium currents, and persists in sodium-free solution, or when calcium is replaced with strontium or barium. Our results support the hypothesis of a purely calcium-dependent impulse.