Molecular engineering aspects for the production of new and modified biosurfactants.

Biosurfactants are of considerable industrial value as their high tenside activity in combination with their biocompatibility makes them attractive for many applications. In particular members of the lipopeptide family of biosurfactants contain significant potentials for the pharmaceutical industry due to their intrinsic antibiotic characteristics. The high frequency of lipopeptide (LP) production in common soil microorganisms in combination with the enormous structural diversity of the synthesized biosurfactants has created an abundant natural pool of compounds with potentially interesting properties. Unfortunately, the bioactivity of lipopetides against pathogenic microorganisms is often associated with problematic side effects that restrict or even prevent medically relevant applications. The accumulated knowledge of lipopetide biosynthesis and their frequent structural variations caused by natural genetic rearrangements has therefore motivated numerous approaches in order to manipulate biosurfactant composition and production mechanisms. This chapter will give an overview on current engineering strategies that aim to obtain lipopeptide biosurfactants with redesigned structures and optimized properties.

Manipulation of thiocillin variants by prepeptide gene replacement: structure, conformation, and activity of heterocycle substitution mutants.

Bacillus cereus ATCC 14579 converts the C-terminal 14 residues of a 52-mer prepeptide into a related set of eight variants of the thiocillin subclass of thiazolyl peptide antibiotics by a cascade of post-translational modifications that alter 13 of those 14 residues. We have introduced prepeptide gene variants into a knockout strain to conduct an alanine scan of all 14 progenitor residues, as well as a serine scan of the six cysteine residues that are converted to thiazoles in the mature natural product. No mature scaffolds were detected for the S1A and S10A mutants, consistent with their roles as the source of the pyridine core. In both the alanine and serine scans, only one substitution mutant failed to produce a mature scaffold: cysteine 11. Cysteine to serine mutants gave mixture of dehydrations, aromatizations, and unaltered alcohol side chains depending on position. Overall, substitutions that altered the trithiazolylpyridine core or reduced the conformational rigidity of the 26-membered macrocyclic loop led to loss of antibiotic activity. In total, 21 peptide mutants were cultured, from which production of 107 compounds was observed and 94 compounds, representing 17 structural mutants, were assayed for antibiotic activity. High-resolution NMR solution structures were determined for one mutant and one wild-type compound. These structures demonstrate that the tight conformational rigidity of the natural product is severely disrupted by loss of even a single heterocycle, perhaps accounting for the attendant loss of activity in such mutants.

Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in (15)N-(13)C-ILV methyl labeled proteins.

We present a time-shared 3D HSQC-NOESY experiment that enables one to simultaneously record (13)C- and (15)N-dispersed spectra in Ile, Leu and Val (ILV) methyl-labeled samples. This experiment is designed to delineate the two spectra which would otherwise overlap with one another when acquired together. These spectra display nOe correlations in the detected proton dimension, i.e. with maximum resolution. This is in contrast to NOESY-HSQC types of experiments that provide cross-peaks in the indirect dimension with low resolution due to limits in experimental time. The technique is particularly advantageous at high field where even longer experimental times would be required for comparable resolution in NOESY-HSQC experiments. The method is demonstrated at 900 MHz and at 750 MHz on 37 and 31 kDa proteins, respectively. The resolution and time saving provided in this experiment was crucial for solving the structures of these two proteins.

A double TROSY hNCAnH experiment for efficient assignment of large and challenging proteins.

We present an experiment that allows for a straightforward assignment of NMR resonances, even in large and/or challenging proteins. A single 3D double-TROSY experiment provides three pairs of sequential correlations between two alpha carbons, two amide protons, and two nitrogen nuclei. Thus, all correlated nuclei can readily be visualized within all dimensions of the resulting spectrum, and chain elongation of sequential amino acids can be effected with this single data set. This resolves ambiguities occurring in traditional methods which involve time-consuming and cumbersome strip comparisons obtained with series of triple-resonance spectra. The experiment makes use of the double TROSY technique to account for signal intensity losses during transfer and evolution periods and was tested on a 500 microM sample of the 33 kDa nonribosomal peptide synthetase protein EntB.

Structural insights into nonribosomal peptide enzymatic assembly lines.

Nonribosomal peptides have a variety of medicinal activities including activity as antibiotics, antitumor drugs, immunosuppressives, and toxins. Their biosynthesis on multimodular assembly lines as a series of covalently tethered thioesters, in turn covalently attached on pantetheinyl arms on carrier protein way stations, reflects similar chemical logic and protein machinery to fatty acid and polyketide biosynthesis. While structural information on excised or isolated catalytic adenylation (A), condensation (C), peptidyl carrier protein (PCP) and thioesterase (TE) domains had been gathered over the past decade, little was known about how the NRPS catalytic and carrier domains interact with each other both within and across elongation or termination modules. This Highlight reviews recent breakthrough achievements in both X-ray and NMR spectroscopic studies that illuminate the architecture of NRPS PCP domains, PCP-containing didomain-fragments and of a full termination module (C-A-PCP-TE).

Cascade reactions during coronafacic acid biosynthesis: elongation, cyclization, and functionalization during Cfa7-catalyzed condensation.

Herein, the biogenesis of the hydrindane ring system within coronafacic acid (CFA) has been investigated. These studies reveal that in addition to the canonical polyketide chain elongation and functionalization encoded by type I polyketide synthase (PKSs), cascade reactions can take place during assembly line-like biosynthesis. Indeed, upon Cfa7-catalyzed Claisen condensation between enzyme-bound malonate and an N-acetylcysteamine (SNAC) thioester, latent reactivity within the elongated enzyme-bound intermediate is unveiled. This reactivity translates into an intramolecular cyclization, which can proceed in a facile manner as observed by the enzyme-independent cyclization of a linear beta-ketothioester intermediate.

beta-Hydroxylation of the aspartyl residue in the phytotoxin syringomycin E: characterization of two candidate hydroxylases AspH and SyrP in Pseudomonas syringae.

The pseudomonal phytotoxin syringomycin E and related nonribosomal peptides contain an L- threo-beta-hydroxyaspartyl residue at the eighth position of the lipodepsipeptide backbone as part of a conserved nonproteinogenic tripeptide motif. Informatic analysis of the P. syringae genome suggests only one putative non-heme iron hydroxylase, AspH. On heterologous expression in Escherichia coli AspH shows robust catalytic activity with free L-Asp and L-Asp thioesters to make beta-OH-Asp but yields the erythro diastereomer rather than the threo configuration that is found in syringomycin. Further analysis of the Syr gene cluster indicated that SyrP, previously annotated as the gene regulatory protein for the five-gene Syr cluster, is actually homologous to the known non-heme mononuclear iron hydroxylase TauD. Indeed, purified SyrP acts on Asp tethered as the protein-bound S-pantetheinyl thioester on the eighth module of the SyrE megasynthetase. The hydroxylation gives the anticipated L- threo-3-OH-Asp diastereomer found in syringomycin. The knockout of syrP abolishes the production of the mature syringomycin E, while knockout of aspH has no effect on syringomycin production.

Dynamic thiolation-thioesterase structure of a non-ribosomal peptide synthetase.

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations. In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation-thioesterase (T-TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4'-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T-TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T-TE interaction described here provides a model for NRPS, PKS and FAS function in general as T-TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.

Structural basis for the selectivity of the external thioesterase of the surfactin synthetase.

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds. Type I thioesterases (TEI) catalyse the release step from the assembly line of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4'-phosphopantetheine (4'-PP) cofactor that is covalently attached to thiolation (T) domains. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4'-PP cofactors of holo-T domains of NRPS and PKS systems. Mispriming of 4'-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4'-PP cofactor, is acetylated in bacteria. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.

An eight residue fragment of an acyl carrier protein suffices for post-translational introduction of fluorescent pantetheinyl arms in protein modification in vitro and in vivo.

Genetically encoded tags for tracking a given protein continue to be of great interest in a multitude of in vitro and in vivo contexts. Acyl carrier proteins, both free-standing and as embedded 80-100 residue domains, contain a specific serine side chain that undergoes post-translational pantetheinylation from CoASH as donor substrate. We have previously used phage display methods to select a 12 residue fragment that retains recognition for modification by the Escherichia coli phosphopantetheinyltransferase (PPTase) AcpS. In this work, we have used (15)N-HSQC based NMR titration experiments of a 12-residue peptide substrate with AcpS to identify six specifically interacting residues (S3, L4, D5, M6, W9, and L11) without the formation of any notable secondary structure. Synthesis of a corresponding octapeptide containing 5 of the 6 interacting residues generated a minimal fragment capable of efficient post-translational phosphopantetheinylation. Genetic insertion of this eight residue coding sequence into the proteins sonic hedgehog and transferrin receptor enabled good in vitro and in vivo PPTase-mediated modification by a series of fluorescent CoAs, leading to a set of fluorescent proteins with a peptide tag minimally perturbant to protein folds.

Biosynthetic tailoring of microcin E492m: post-translational modification affords an antibacterial siderophore-peptide conjugate.

The present work reveals that four proteins, MceCDIJ, encoded by the MccE492 gene cluster are responsible for the remarkable post-translational tailoring of microcin E492 (MccE492), an 84-residue protein toxin secreted by Klebsiella pneumonaie RYC492 that targets neighboring Gram-negative species. This modification results in attachment of a linearized and monoglycosylated derivative of enterobactin, a nonribosomal peptide and iron scavenger (siderophore), to the MccE492m C-terminus. MceC and MceD derivatize enterobactin by C-glycosylation at the C5 position of a N-(2,3-dihydroxybenzoyl)serine (DHB-Ser) moiety and regiospecific hydrolysis of an ester linkage in the trilactone scaffold, respectively. MceI and MceJ form a protein complex that attaches C-glycosylated enterobactins to the C-terminal serine residue of both a C10 model peptide and full-length MccE492. In the enzymatic product, the C-terminal serine residue is covalently attached to the C4' oxygen of the glucose moiety. Nonenzymatic and base-catalyzed migration of the peptide to the C6' position affords the C6' glycosyl ester linkage observed in the mature toxin, MccE492m, isolated from bacterial cultures.

Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases.

The flavin-dependent halogenase RebH catalyzes the formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin. The reaction of FADH2, Cl-, and O2 in the active site generates the powerful oxidant HOCl, which was presumed to carry out the chlorination reaction. Herein, we demonstrate the formation of a long-lived chlorinating intermediate (t1/2 = 63 h at 4 degrees C) when RebH, FADH2, Cl-, and O2 react in the absence of substrate tryptophan. This intermediate remained on the enzyme after removal of FAD and transferred chlorine to tryptophan with kinetically competent rates. The identity of this intermediate is suggested by the X-ray crystal structure of RebH, which revealed an active site Lys79 located in a central position between flavin and tryptophan binding sites and just 4.1 A above C7 of tryptophan. The chlorinating species is proposed to be a Lys-epsilonNH-Cl (lysine chloramine) from reaction of enzyme-generated HOCl with the active site Lys79. This covalent enzyme chloramine likely plays a key role in directing regiospecific chlorination of substrate in this important class of biosynthetic enzymes.

Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies.

Cell-free expression techniques have emerged as promising tools for the production of membrane proteins for structural and functional analysis. Elimination of toxic effects and a variety of options to stabilize the synthesized proteins enable the synthesis of otherwise difficult to obtain proteins. Modifications in the reaction design result in preparative scale production rates of cell-free reactions and yield in milligram amounts of membrane proteins per one millilitre of reaction volume. A diverse selection of detergents can be supplied into the reaction system without inhibitory effects to the translation machinery. This offers the unique opportunity to produce a membrane protein directly into micelles of a detergent of choice. We present detailed protocols for the cell-free production of membrane proteins in different modes and we summarize the current knowledge of this technique. A special emphasize will be on the production of soluble and functionally folded membrane proteins in presence of suitable detergents. In addition, we will highlight the advantages of cell-free expression for the structural analysis of membrane proteins especially by liquid state nuclear magnetic resonance spectroscopy and we will discuss new strategies for structural approaches.

Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris.

The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 A resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.

Structure of Aquifex aeolicus argonaute highlights conformational flexibility of the PAZ domain as a potential regulator of RNA-induced silencing complex function.

Gene silencing mediated by RNA interference requires the sequence-specific recognition of target mRNA by the endonuclease Argonaute, the primary enzymatic component of the RNA-induced silencing complex. We report the crystal structure of Aquifex aeolicus Argonaute, refined at 3.2A resolution. Relative to recent Argonaute structures, a 24 degrees reorientation of the PAZ domain in our structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. This rearrangement leads to a branched, Y-shaped system of grooves that extends through the molecule and merges in a central channel containing the catalytic residues. A 5.5-ns molecular dynamics simulation of Argonaute shows a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute monitored by total internal reflection fluorescence spectroscopy shows biphasic kinetics, also indicative of domain rearrangement upon DNA binding. Conformational rearrangement of the PAZ domain may therefore be critical for the catalytic cycle of Argonaute and the RNA-induced silencing complex.

Carrier protein structure and recognition in polyketide and nonribosomal peptide biosynthesis.

Carrier proteins, 80-100 residues in length, serve as information-rich platforms to present growing acyl and peptidyl chains as covalently tethered phosphopantetheinyl-thioester intermediates during the biosynthesis of fatty acid, polyketide, and nonribosomal natural products. Carrier proteins are recognized both in cis and in trans by partner catalytic domains that effect chain-elongating condensations, redox adjustments, other tailoring steps, and finally kinetically controlled disconnection and release of the mature natural product. Dissection of regions of carrier proteins that are specifically recognized by upstream and downstream catalytic partner proteins is deciphering the logic for multiprotein assembly line construction of these large classes of natural products.

A new structural domain in the Escherichia coli RcsC hybrid sensor kinase connects histidine kinase and phosphoreceiver domains.

The Rcs signalling pathway controls a variety of physiological functions like capsule synthesis, cell division or motility in prokaryotes. The Rcs regulation cascade, involving a multi-step phosphorelay between the two membrane-bound hybrid sensor kinases RcsC and RcsD and the global regulator RcsB, is, up to now, one of the most complicated regulatory systems in bacteria. To understand the structural basis of Rcs signal transduction, NMR spectroscopy was employed to determine the solution structure of the RcsC C terminus, possessing a phosphoreceiver domain (RcsC-PR), and a region previously described as a long linker between the histidine kinase domain of RcsC (RcsC-HK) and the RcsC-PR. We have found that the linker region comprises an independent structural domain of a new alpha/beta organization, which we named RcsC-ABL domain (Alpha/Beta/Loop). The ABL domain appears to be a conserved and unique structural element of RcsC-like kinases with no significant sequence homology to other proteins. The second domain of the C terminus, the RcsC-PR domain, represents a well-folded CheY-like phosphoreceiver domain with the central parallel beta-sheet covered with two alpha-helical layers on both sides. We have mapped the interaction of RcsC-ABL and RcsC-PR with the histidine phosphotransfer domain (HPt) of RcsD. In addition we have characterized the interaction with and the conformational effects of Mg2+ and the phosphorylation mimetic BeF(-)(3) on RcsC-ABL and RcsC-PR.

Combination of cell-free expression and NMR spectroscopy as a new approach for structural investigation of membrane proteins.

Despite major technical advance in methods used for structural investigations of proteins structure determination of membrane proteins still poses a significant challenge. Recently, the application of cell-free expression systems to membrane proteins has demonstrated that this technique can be used to produce quantities sufficient for structural investigations for many different membrane proteins. In particular for NMR spectroscopy, cell-free expression provides major advantages since it allows for amino acid type selective and even amino acid position specific labeling. In this mini-review we discuss the combination of cell-free membrane protein expression and liquid state NMR spectroscopy.

Conformational switches modulate protein interactions in peptide antibiotic synthetases.

Protein dynamics plays an important role in protein function. Many functionally important motions occur on the microsecond and low millisecond time scale and can be characterized by nuclear magnetic resonance relaxation experiments. We describe the different states of a peptidyl carrier protein (PCP) that play a crucial role in its function as a peptide shuttle in the nonribosomal peptide synthetases of the tyrocidine A system. Both apo-PCP (without the bound 4'-phosphopantetheine cofactor) and holo-PCP exist in two different stable conformations. We show that one of the apo conformations and one of the holo conformations are identical, whereas the two remaining conformations are only detectable by nuclear magnetic resonance spectroscopy in either the apo or holo form. We further demonstrate that this conformational diversity is an essential prerequisite for the directed movement of the 4'-PP cofactor and its interaction with externally acting proteins such as thioesterases and 4'-PP transferase.