Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Convergent evolution of bacterial ceramide synthesis.

The bacterial domain produces numerous types of sphingolipids with various physiological functions. In the human microbiome, commensal and pathogenic bacteria use these lipids to modulate the host inflammatory system. Despite their growing importance, their biosynthetic pathway remains undefined since several key eukaryotic ceramide synthesis enzymes have no bacterial homolog. Here we used genomic and biochemical approaches to identify six proteins comprising the complete pathway for bacterial ceramide synthesis. Bioinformatic analyses revealed the widespread potential for bacterial ceramide synthesis leading to our discovery of a Gram-positive species that produces ceramides. Biochemical evidence demonstrated that the bacterial pathway operates in a different order from that in eukaryotes. Furthermore, phylogenetic analyses support the hypothesis that the bacterial and eukaryotic ceramide pathways evolved independently.

Developing deprotectase biocatalysts for synthesis.

Organic synthesis often requires multiple steps where a functional group (FG) is concealed from reaction by a protecting group (PG). Common PGs include N-carbobenzyloxy (Cbz or Z) of amines and tert-butyloxycarbonyl (OtBu) of acids. An essential step is the removal of the PG, but this often requires excess reagents, extensive time and can have low % yield. An overarching goal of biocatalysis is to use "green" or "enzymatic" methods to catalyse chemical transformations. One under-utilised approach is the use of "deprotectase" biocatalysts to selectively remove PGs from various organic substrates. The advantage of this methodology is the exquisite selectivity of the biocatalyst to only act on its target, leaving other FGs and PGs untouched. A number of deprotectase biocatalysts have been reported but they are not commonly used in mainstream synthetic routes. This study describes the construction of a cascade to deprotect doubly-protected amino acids. The well known Bacillus BS2 esterase was used to remove the OtBu PG from various amino acid substrates. The more obscure Sphingomonas Cbz-ase (amidohydrolase) was screened with a range of N-Cbz-modified amino acid substrates. We then combined both the BS2 and Cbz-ase together for a 1 pot, 2 step deprotection of the model substrate CBz-L-Phe OtBu to produce the free L-Phe. We also provide some insight into the residues involved in substrate recognition and catalysis using docked ligands in the crystal structure of BS2. Similarly, a structural model of the Cbz-ase identifies a potential di-metal binding site and reveals conserved active site residues. This new biocatalytic cascade should be further explored for its application in chemical synthesis.

Creation of an Engineered Amide Synthetase Biocatalyst by the Rational Separation of a Two-Step Nitrile Synthetase.

The synthesis of amides through acid and amine coupling is one of the most commonly used reactions in medicinal chemistry, yet still requires atom-inefficient coupling reagents. There is a current demand to develop greener, biocatalytic approaches to amide bond formation. The nitrile synthetase (NS) enzymes are a small family of ATP-dependent enzymes which catalyse the transformation of a carboxylic acid into the corresponding nitrile via an amide intermediate. The Bacillus subtilis QueC (BsQueC) is an NS involved in the synthesis of 7-cyano-7-deazaguanine (CDG) natural products. Through sequence homology and structural analysis of BsQueC we identified three highly conserved residues, which could potentially play important roles in NS substrate binding and catalysis. Rational engineering led to the creation of a NS K163A/R204A biocatalyst that converts the CDG acid into the primary amide, but does not proceed to the nitrile. This study suggests that NSs could be further developed for coupling agent-free, amide-forming biocatalysts.

BioWF: A Naturally-Fused, Di-Domain Biocatalyst from Biotin Biosynthesis Displays an Unexpectedly Broad Substrate Scope.

The carbon backbone of biotin is constructed from the C7 di-acid pimelate, which is converted to an acyl-CoA thioester by an ATP-dependent, pimeloyl-CoA synthetase (PCAS, encoded by BioW). The acyl-thioester is condensed with ʟ-alanine in a decarboxylative, Claisen-like reaction to form an aminoketone (8-amino-7-oxononanoic acid, AON). This step is catalysed by the pyridoxal 5'-phosphate (PLP)-dependent enzyme (AON synthase, AONS, encoded by BioF). Distinct versions of Bacillus subtilis BioW (BsBioW) and E. coli BioF (EcBioF) display strict substrate specificity. In contrast, a BioW-BioF fusion from Corynebacterium amycolatum (CaBioWF) accepts a wider range of mono- and di-fatty acids. Analysis of the active site of the BsBioW : pimeloyl-adenylate complex suggested a key role for a Phe (F192) residue in the CaBioW domain; a F192Y mutant restored the substrate specificity to pimelate. This surprising substrate flexibility also extends to the CaBioF domain, which accepts ʟ-alanine, ʟ-serine and glycine. Structural models of the CaBioWF fusion provide insight into how both domains interact with each other and suggest the presence of an intra-domain tunnel. The CaBioWF fusion catalyses conversion of various fatty acids and amino acids to a range of AON derivatives. Such unexpected, natural broad substrate scope suggests that the CaBioWF fusion is a versatile biocatalyst that can be used to prepare a number of aminoketone analogues.

Solution Structure and Conformational Dynamics of a Doublet Acyl Carrier Protein from Prodigiosin Biosynthesis.

The acyl carrier protein (ACP) is an indispensable component of both fatty acid and polyketide synthases and is primarily responsible for delivering acyl intermediates to enzymatic partners. At present, increasing numbers of multidomain ACPs have been discovered with roles in molecular recognition of trans-acting enzymatic partners as well as increasing metabolic flux. Further structural information is required to provide insight into their function, yet to date, the only high-resolution structure of this class to be determined is that of the doublet ACP (two continuous ACP domains) from mupirocin synthase. Here we report the solution nuclear magnetic resonance (NMR) structure of the doublet ACP domains from PigH (PigH ACP1-ACP2), which is an enzyme that catalyzes the formation of the bipyrrolic intermediate of prodigiosin, a potent anticancer compound with a variety of biological activities. The PigH ACP1-ACP2 structure shows each ACP domain consists of three conserved helices connected by a linker that is partially restricted by interactions with the ACP1 domain. Analysis of the holo (4'-phosphopantetheine, 4'-PP) form of PigH ACP1-ACP2 by NMR revealed conformational exchange found predominantly in the ACP2 domain reflecting the inherent plasticity of this ACP. Furthermore, ensemble models obtained from SAXS data reveal two distinct conformers, bent and extended, of both apo (unmodified) and holo PigH ACP1-ACP2 mediated by the central linker. The bent conformer appears to be a result of linker-ACP1 interactions detected by NMR and might be important for intradomain communication during the biosynthesis. These results provide new insights into the behavior of the interdomain linker of multiple ACP domains that may modulate protein-protein interactions. This is likely to become an increasingly important consideration for metabolic engineering in prodigiosin and other related biosynthetic pathways.

Direct monitoring of biocatalytic deacetylation of amino acid substrates by 1H NMR reveals fine details of substrate specificity.

Amino acids are key synthetic building blocks that can be prepared in an enantiopure form by biocatalytic methods. We show that the l-selective ornithine deacetylase ArgE catalyses hydrolysis of a wide-range of N-acyl-amino acid substrates. This activity was revealed by 1H NMR spectroscopy that monitored the appearance of the well resolved signal of the acetate product. Furthermore, the assay was used to probe the subtle structural selectivity of the biocatalyst using a substrate that could adopt different rotameric conformations.

Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase.

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.

Using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters.

Biotin is an essential vitamin in plants and mammals, functioning as the carbon dioxide carrier within central lipid metabolism. Bacterial pimeloyl-CoA synthetase (BioW) acts as a highly specific substrate-selection gate, ensuring the integrity of the carbon chain in biotin synthesis. BioW catalyzes the condensation of pimelic acid (C7 dicarboxylic acid) with CoASH in an ATP-dependent manner to form pimeloyl-CoA, the first dedicated biotin building block. Multiple structures of Bacillus subtilis BioW together capture all three substrates, as well as the intermediate pimeloyl-adenylate and product pyrophosphate (PPi), indicating that the enzyme uses an internal ruler to select the correct dicarboxylic acid substrate. Both the catalytic mechanism and the surprising stability of the adenylate intermediate were rationalized through site-directed mutagenesis. Building on this understanding, BioW was engineered to synthesize high-value heptanoyl (C7) and octanoyl (C8) monocarboxylic acid-CoA and C8 dicarboxylic-CoA products, highlighting the enzyme's synthetic potential.

Sphingolipid biosynthesis in man and microbes.

A new review covering up to 2018 Sphingolipids are essential molecules that, despite their long history, are still stimulating interest today. The reasons for this are that, as well as playing structural roles within cell membranes, they have also been shown to perform a myriad of cell signalling functions vital to the correct function of eukaryotic and prokaryotic organisms. Indeed, sphingolipid disregulation that alters the tightly-controlled balance of these key lipids has been closely linked to a number of diseases such as diabetes, asthma and various neuropathologies. Sphingolipid biogenesis, metabolism and regulation is mediated by a large number of enzymes, proteins and second messengers. There appears to be a core pathway common to all sphingolipid-producing organisms but recent studies have begun to dissect out important, species-specific differences. Many of these have only recently been discovered and in most cases the molecular and biochemical details are only beginning to emerge. Where there is a direct link from classic biochemistry to clinical symptoms, a number a drug companies have undertaken a medicinal chemistry campaign to try to deliver a therapeutic intervention to alleviate a number of diseases. Where appropriate, we highlight targets where natural products have been exploited as useful tools. Taking all these aspects into account this review covers the structural, mechanistic and regulatory features of sphingolipid biosynthetic and metabolic enzymes.

Non-invasive 19F NMR analysis of a protein-templated N-acylhydrazone dynamic combinatorial library.

Dynamic combinatorial chemistry (DCC) is a powerful tool to identify ligands for biological targets. We used 19F NMR as an in situ, non-invasive technique for measuring the composition of a dynamic combinatorial library (DCL) of N-acylhydrazones (NAHs). An NAH DCL, constructed from a fluoro-aromatic aldehyde and a small set of hydrazides, was targetted at ecFabH, an essential enzyme in bacterial fatty acid biosynthesis. Our NMR analysis identified a tert-butyl NAH as the best binder which was confirmed by enzymatic assay.

The carbon chain-selective adenylation enzyme TamA: the missing link between fatty acid and pyrrole natural product biosynthesis.

The marine bacterium Pseudoalteromonas tunicata produces the bipyrrole antibiotic tambjamine YP1. This natural product is built from common amino acid and fatty acid building blocks in a biosynthetic pathway that is encoded in the tam operon which contains 19 genes. The exact role that each of these Tam proteins plays in tambjamine biosynthesis is not known. Here, we provide evidence that TamA initiates the synthesis and controls the chain length of the essential tambjamine fatty amine tail. Sequence analysis suggests the unusual TamA is comprised of an N-terminal adenylation (ANL) domain fused to a C-terminal acyl carrier protein (ACP). Mass spectrometry analysis of recombinant TamA revealed the surprising presence of bound C11 and C12 acyl-adenylate intermediates. Acylation of the ACP domain was observed upon attachment of the phosphopantetheine (4'-PP) arm to the ACP. We also show that TamA can transfer fatty acids ranging in chain length from C6-C13 to an isolated ACP domain. Thus TamA bridges the gap between primary and secondary metabolism by linking fatty acid and pyrrole biosynthetic pathways.

Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei.

Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.

Characterization of secreted sphingosine-1-phosphate lyases required for virulence and intracellular survival of Burkholderia pseudomallei.

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein, we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL-deficient yeast strain and conferred resistance to the antimicrobial sphingolipid D-erythro-sphingosine. We report that secretion of these bacterial S1PLs is pH-dependent, and is observed during intracellular infection. S1PL-deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia-infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP-1 and reduced their intracellular survival. In summary, our studies confirm bacterial-encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host-pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection.

Structural evidence for the covalent modification of FabH by 4,5-dichloro-1,2-dithiol-3-one (HR45).

We use mass spectrometry analysis and molecular modelling to show the established antimicrobial inhibitor 4,5-dichloro-1,2-dithiol-3-one (HR45) acts by forming a covalent adduct with the target ß-ketoacyl-ACP synthase III (FabH). The 5-chloro substituent directs attack of the essential active site thiol (C112) via a Michael-type addition elimination reaction mechanism.

Determination of Protein Thiol Reduction Potential by Isotope Labeling and Intact Mass Measurement.

Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential (E(⊕)) for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods, but top-down mass spectrometry recently has been shown to enable the quantification of E(⊕) for thiol oxidations. In this paper, we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure E(⊕) for the thiols in both E. coli Thioredoxin 1 and human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using liquid chromatography-mass spectroscopy (LC-MS) analyses and does not necessarily require top-down fragmentation. In addition to analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E(⊕) measurements in samples that contain mixtures of proteins. Thus, the combination of experimential methodology and data analysis regime has the potential to make such measurements in a high-throughput manner and in a manner that is more accessible to a broad community of protein scientists.

Continuous colorimetric assay that enables high-throughput screening of N-acetylamino acid racemases.

N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology interest. In order to identify novel activities and to improve these enzymes by engineering approaches, suitable screening methods are necessary. Previous engineering of the NAAAR from Amycolatopsis Ts-1-60 was achieved by relying on an in vivo selection system that linked the viability of an E. coli L-methionine auxotroph to the activity of the improved enzyme. However, this assay was only suitable for the screening of N-acetyl-D-methionine, therefore limiting the potential to evolve this enzyme toward other natural or non-natural acetylated amino acids. Here, we report the optimization and application of a spectrophotometric microtiter-plate-based assay for NAAAR. The assay is based on the detection of the amino acid reaction product formed by hydrolysis of the N-acylated substrate by an L-amino acid acylase and its subsequent oxidation by an FAD-dependent L-amino acid oxidase (L-AAO). Cofactor recycling of the L-AAO leads to the formation of hydrogen peroxide which is easily monitored using horseradish peroxidase (HRP) and o-dianisidine. This method allowed for the determination of the kinetic parameters of NAAAR and led to the identification of N-acetyl-D-naphthylalanine as a novel NAAAR substrate. This robust method is also suitable for the high-throughput screening of NAAAR mutant gene libraries directly from cell lysates.

Desalting large protein complexes during native electrospray mass spectrometry by addition of amino acids to the working solution.

Native mass spectrometry is a rapidly emerging field for characterising the structure of proteins and protein assemblies. The technique relies on electrospray ionisation (ESI) to efficiently ionise the protein analyte and transmit it into the gas phase with retention of protein structure, non-covalent protein-ligand and protein-protein interactions. In native ESI, both the ionisation efficiency and the resulting mass spectral signal is adversely effected by the presence of non-volatile inorganic salts, such as sodium chloride, which form extensive adducts with the protein ions. Consequently, there is great interest in finding experimental strategies that mitigate these phenomena. Here we report our findings that the addition of 10 mM l-serine to the ESI spray solution reduces the adverse effects of sodium adduction to proteins. In the analysis of bovine serum albumin (BSA; 66 kDa), 10 mM serine increased signal to noise ratio (S/N) ∼4 fold. This increase in sensitivity was accompanied by peak narrowing (∼10 fold), which allowed more precise assignment of molecular mass. Similar effects were observed when analysing protein complexes - serine palmitoyl transferase (SPT, a 92 kDa homodimer), enolase (a 93 kDa homodimer); and alcohol dehydrogenase (ADH, a 148 kDa tetramer). Reduction in sodium ion adduction occurs with no loss of the non-covalent protein-protein interactions, and with little effect on the overall observed charge state-distribution. As a consequence of increasing signal intensity, the addition of serine to the ESI spray solution greatly improved the quality of the data obtained from native top-down electron-capture dissociation (ECD) experiments. In ECD analysis of native BSA, we observed an increasing in the S/N of all ECD fragments upon addition of 10 mM l-serine. The number of ECD fragments we observed with S/N > 1.5 increased from 15 to 44 and the number of assigned c and z ions increased from 5 to 16. Finally we show that this phenomenon is not specific to l-serine, and occurs with several amino acids such as l-alanine. Our findings suggest that desalting may occur via binding of sodium ion to the amino acid in solution. This simple and inexpensive strategy has broad utility for improving the mass spectra obtained in a range of MS-based structural proteomic studies.

Collaborative regulation of yeast SPT-Orm2 complex by phosphorylation and ceramide.

The homeostatic regulation of serine palmitoyltransferase (SPT) activity in yeast involves N-terminal phosphorylation of Orm proteins, while higher eukaryotes lack these phosphorylation sites. Although recent studies have indicated a conserved ceramide-mediated feedback inhibition of the SPT-ORM/ORMDL complex in higher eukaryotes, its conservation and relationship with phosphorylation regulation in yeast remain unclear. Here, we determine the structure of the yeast SPT-Orm2 complex in a dephosphomimetic state and identify an evolutionarily conserved ceramide-sensing site. Ceramide stabilizes the dephosphomimetic Orm2 in an inhibitory conformation, facilitated by an intramolecular ß-sheet between the N- and C-terminal segments of Orm2. Moreover, we find that a phosphomimetic mutant of Orm2, positioned adjacent to its intramolecular ß-sheet, destabilizes the inhibitory conformation of Orm2. Taken together, our findings suggest that both Orm dephosphorylation and ceramide binding are crucial for suppressing SPT activity in yeast. This highlights a distinctive regulatory mechanism in yeast involving the collaborative actions of phosphorylation and ceramide.