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bioRxiv2018

Fusion surface structure, function, and dynamics of gamete fusogen HAP2

HAP2 is a class II gamete fusogen in many eukaryotic kingdoms. A crystal structure of Chlamydomonas HAP2 shows a trimeric fusion state. Domains D1, D2.1 and D2.2 line the 3-fold axis; D3 and a stem pack against the outer surface. Surprisingly, hydrogen-deuterium exchange shows that surfaces of D1, D2.2 and D3 closest to the 3-fold axis are more dynamic than exposed surfaces. Three fusion helices in the fusion loop of each monomer expose hydrophobic residues at the trimer apex that are splayed from the 3-fold axis, leaving a solvent-filled cavity between the fusion loops in each monomer. At the base of the long fusion loop, Arg185 docks in a carbonyl cage. Comparisons to other structures, dynamics, and the greater effect on Chlamydomonas gamete fusion of mutation of axis-proximal than axis-distal fusion helices suggest that the apical portion of each monomer could tilt toward the 3-fold axis with merger of the fusion helices into a common fusion surface.

biochemistry↗

bioRxiv2018

Cis-nonPro Peptides: Genuine Occurrences and their Functional Roles

While cis peptides preceding proline can occur about 5% of the time, cis peptides preceding any other residue ("cis-nonPro" peptides) are an extremely rare feature in protein structures, of considerable importance for two opposite reasons. On one hand, their genuine occurrences are mostly found at sites critical to biological function, from the active sites of carbohydrate enzymes to rare adjacent-residue disulfide bonds. On the other hand, a cis-nonPro can easily be misfit into weak or ambiguous electron density, which led to a high incidence of unjustified cis-nonPro over the 2006-2015 decade. This paper uses high-resolution crystallographic data and especially stringent quality-filtering at the residue level to identify genuine occurrences of cis-nonPro, and to survey both individual examples and broad patterns of their functionality. We explain the procedure developed to identify genuine cis-nonPro examples with almost no false positives. We then survey a large sample of the varied functional roles and structural contexts of cis-nonPro, including the uses of specific amino acids for particular purposes. We emphasize aspects not previously covered: that cis-nonPro always (except for vicinal disulfides) occur in highly ordered structure, and especially the great concentration of occurrence in proteins that process or bind carbohydrates (identified by occurrence on the CAZy website).

biochemistry↗

bioRxiv2018

Enhanced inhibition of influenza virus infection by peptide-noble metal nanoparticle conjugates

Influenza virus is a major medical and veterinary health concern and causes global pandemics. The peptide FluPep is an established inhibitor of influenza virus infectivity in model systems. We have explored the potential for FluPep functionalised noble metal nanoparticle to enhance the antiviral activity of the peptide Flupep and determined their potential for the delivery of FluPep. The FluPep ligand designed here is FluPep extended at its N-terminus with the sequence CVVVTAAA-, to allow its incorporation into a mix matrix ligand shell of a peptidol and an alkanethiol ethyleneglycol comprising 70% H-CVVVTol and 30% HS(CH2)11(ethyleneglycol)4 (both mole/mole). Gold and silver nanoparticles (~10 nm diameter) prepared with up to 5% (mole/mole) FluPep ligand contained in the mixture of mix-matrix peptide ligands remained as stable as the control mix-matrix coated nanoparticles against ligand exchange with dithiothreitol. FluPep ligand was found to inhibit viral plaque formation in canine MDCK cells (IC50 2.1 nM), but was less potent than FluPep itself (IC50 140 pM). FluPep ligand functonalised nanoparticles retained antiviral activity in the plaque assay. Moreover, at low grafting densities (where nanoparticles incorporate ~1 FluPep ligand, the antiviral potency in terms of FluPep ligand concentration was enhanced significantly for gold and silver nanoparticles (IC50 ~8-fold and ~3-fold lower, respectively). At higher grafting density the potency relative to free FluPep ligand concentration decreased. The data demonstrate that conjugation of FluPep to gold and silver nanoparticles enhances its antiviral potency; the antimicrobial activity of silver ions may enable the design of even more potent anti-microbial inhibitors.

biochemistry↗

bioRxiv2018

Characterization of novel inhibition of indoleamine 2,3-dioxygenase by targeting its apo form.

Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the rate-limiting step in the kynurenine pathway of tryptophan (TRP) metabolism. As an inflammation-induced immunoregulatory enzyme, pharmacological inhibition of IDO1 activity is currently being pursued as a potential therapeutic tool for the treatment of cancer and other disease states. As such, a detailed understanding of the mechanism of action of established and novel IDO1 inhibitors remains of great interest. Comparison of a newly-developed IDO1 inhibitor (GSK5628) to the existing best-in-class compound, epacadostat (Incyte), allows us to report on a unique inhibition mechanism for IDO1. Here, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme-free conformation of the enzyme (apo-IDO1) while epacadostat coordinates its binding with the iron atom of the IDO1 heme cofactor. Comparison of these two compounds in cellular systems reveals a long-lasting inhibitory effect of GSK5628, undescribed for other known IDO1 inhibitors. Detailed characterization of this apo-binding mechanism for IDO1 inhibition may help design superior inhibitors or may confer a unique competitive advantage over other IDO1 inhibitors vis-a-vis specificity and pharmacokinetic parameters.

biochemistry↗

bioRxiv2018

Tau monomer encodes strains

Tauopathies have diverse presentation, progression, and neuropathology. They are linked to tau prion strains, self-replicating assemblies of unique quaternary conformation. Strains can be propagated indefinitely in cultured cells, and induce unique patterns of transmissible neuropathology upon inoculation into mice. Aggregates from a single strain reproduce only that strain upon re-inoculation into cells or mice. DS9 and DS10 cell lines propagate distinct synthetic strains. Surprisingly, DS9 monomer inoculated into naive cells encoded an identical \"sub-strain,\" whereas DS10 monomer encoded multiple sub-strains. Sub-strains produced distinct pathology upon inoculation into a tauopathy mouse model (PS19). Brain-derived tau monomer from an Alzheimers brain encoded a single strain. Monomer from a corticobasal degeneration brain encoded three sub-strains in which monomer from each encoded all three upon re-inoculation into cells. Tau monomer thus adopts multiple, stable seed-competent conformations, each of which encodes a limited number of strains. This provides insights into the origins of distinct tauopathies.

biochemistry↗

bioRxiv2018

Mammalian stringent-like response mediated by the cytosolic NADPH phosphatase MESH1

Nutrient deprivation triggers stringent response in bacteria, allowing rapid reallocation of resources from proliferation toward stress survival. Critical to this process is the accumulation/degradation of (p)ppGpp regulated by the RelA/SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Therefore, the function of MESH1 remains a mystery. Here, we report that human MESH1 is an efficient cytosolic NADPH phosphatase, an unexpected enzymatic activity that is captured by the crystal structure of the MESH1-NADPH complex. MESH1 depletion promotes cell survival under ferroptosis-inducing conditions by sustaining the level of NADPH, an effect that is reversed by the simultaneous depletion of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Importantly, MESH1 depletion also triggers extensive transcriptional changes that are distinct from the canonical integrated stress response but resemble the bacterial stringent response, implicating MESH1 in a previously uncharacterized stress response in mammalian cells.

biochemistry↗

bioRxiv2018

Proteomic Analysis of NRROS Interactome Reveals the Presence of Chaperones and Mediators of the ERAD Pathway

Negative regulator of reactive oxygen species (NRROS, previously called LRRC33) is a leucine-rich repeat (LRR) domain containing, ER-resident transmembrane protein expressed primarily in lymphoid organs, especially in myeloid cells. We have previously demonstrated that NRROS regulates reactive oxygen species production by phagocytic cells by mediating degradation of NOX2 (gp91phox), a component of NOX2 complex responsible for the oxidative burst in these cells. Since LRR is the only functional domain in NRROS, it is likely to interact with other proteins for its biological functions. Here, by performing immunoprecipitation of NRROS and mass spectrometric analysis, we describe the NRROS interactome in macrophages and demonstrate that NRROS interacts with molecular chaperones/co-chaperones and mediators of the endoplasmic reticulum associated degradation (ERAD) pathway such as calnexin, suggesting a broader role for NRROS in protein biosynthesis and the ER quality control machinery.

biochemistry↗

bioRxiv2018

Mitochondrial ClpX activates an essential metabolic enzyme through partial unfolding

Mitochondria can control the activity, quality, and lifetime of their proteins with their autonomous system of chaperones, but the signals that direct substrate-chaperone interaction and outcome are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting incorporation of cofactor. Here, we ask how unfolding by mtClpX directs activation. We identified sequence and structural features in ALAS that position mtClpX and provide a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding was limited to a subdomain extending from the mtClpX-binding site to the active site. Unfolding along this path was required for mtClpX to gate cofactor access to the ALAS active site. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and suggests how substrate-chaperone interactions can direct the outcome of remodeling.

biochemistry↗

bioRxiv2018

The S. cerevisiae Hrq1 and Pif1 DNA helicases synergistically modulate telomerase activity in vitro

Telomere length homeostasis is vital to maintaining genomic stability and is regulated by multiple factors, including telomerase activity and DNA helicases. The Saccharomyces cerevisiae Pif1 helicase was the first discovered catalytic inhibitor of telomerase, but recent experimental evidence suggests that Hrq1, the yeast homolog of the disease-linked human RecQ-like helicase 4 (RECQL4), plays a similar role via an undefined mechanism. Using yeast extracts enriched for telomerase activity and an in vitro primer extension assay, here we determined the effects of recombinant wild-type and inactive Hrq1 and Pif1 on total telomerase activity and telomerase processivity. We found that titrations of these helicases alone have equal-but-opposite biphasic effects on telomerase, with Hrq1 stimulating activity at high concentrations. When the helicases were combined in reactions, however, they synergistically inhibited or stimulated telomerase activity depending on which helicase was catalytically active. These results suggest that Hrq1 and Pif1 interact and that their concerted activities ensure proper telomere length homeostasis in vivo. We propose a model in which Hrq1 and Pif1 cooperatively contribute to telomere length homeostasis in yeast.

biochemistry↗

bioRxiv2018

N4-acetylcytidine and 5-formylcytidine are present in Saccharomyces cerevisiae mRNAs

Post-transcriptional nucleoside modifications have long been recognized as key modulators of non-coding RNA structure and function. There is an emerging appreciation that the chemical modification of protein-coding messenger RNAs (mRNAs) also plays critical roles in the cell. Although there are over 100 known RNA modifications found in biology only a handful have been identified in mRNAs. We sought to identify and quantify modifications present in the mRNAs of yeast cells using a high throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method that measures the levels of 36 types of RNA nucleosides in parallel. We detected the presence of six modified nucleosides in mRNAs at relatively high abundances: N7-methylguanosine, N6-methyladenosine, 2-O-methylguanosine, 2-O-methylcytosine, N4-acetylcytidine and 5-formylcytidine. Additionally, we investigated how the levels of mRNA modifications vary in response to cellular stress. We find that the concentrations of mRNA modifications including N6-methyladenosine and N4-acetylcytidine change in response to heat stress, glucose starvation and/or oxidative stress. This work expands the repertoire of potential chemical modifications in mRNAs, and utilizes a high-throughput approach to search for modifications that highlights the value of integrating mass-spectrometry tools in the mRNA modification discovery and characterization pipeline.

biochemistry↗

bioRxiv2018

Transition State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present an investigation based on linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs wild type PAS has a much less negative Br0nsted coefficient ({beta}leaving groupobs-Enz = -0.33) than the uncatalyzed reaction ({beta}leavingroupobs = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the TS is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active-site lysine K375, in cooperation with histidine H211. 15N KIEs combined with an increased sensitivity to leaving group ability of the sulfatase activity in neat D2O ({Delta}{beta}leaving groupH-D = +0.06) suggest that the mechanism for S-Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions towards direct proton donation. 18Ononbridge KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate in this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

biochemistry↗

bioRxiv2018

Computational Screening of Potential Inhibitors of Desulfobacter postgatei for Pyrite Scale Prevention in Oil and Gas Wells

Sulfate-reducing bacteria (SRB) such as Desulfobacter postgatei are often found in oil and gas wells. However, they lead to the release of hydrogen sulfide which in turn leads to the formation of iron sulfide scale such as pyrite. ATP sulfurylase is an enzyme present in SRB, which catalyzes the formation of adenylyl sulfate (APS) and inorganic pyrophosphatase (PPi) from ATP and sulfate which is one of the first steps in hydrogen sulfide production by D. postgatei. Virtual screening using molecular docking and machine learning tools was used to identify three potential inhibitors of ATP sulfurylase from a database of about 40 million compounds. These selected hits ((S,E)-1-(4-methoxyphenyl)-3-(9-((m-tolylimino)methyl)-9,10-dihydroanthracen-9-yl)pyrrolidine-2,5-dione;,methyl 2-[[(1S)-5-cyano-2-imino-1-(4-phenylthiazol-2-yl)-3-azaspiro[5.5]undec-4-en-4-yl]sulfanyl]acetate and (4S)-4-(3-chloro-4-hydroxy-phenyl)-1-(6-hydroxypyridazin-3-yl)-3-methyl-4,5-dihydropyrazolo[3,4-b]pyridin-6-ol), which are known as A, B and C respectively) all had good binding affinities with ATP sulfurylase and were further analyzed for their toxicological properties. The molecular docking results showed that all the compounds have negative binding energy with compound A having the highest docking score. However, based on the physicochemical and toxicological properties, compound C is the best choice as it does not violate any of the recommended properties that relate to absorption and distribution. Only compound C was predicted to be both safe and effective as a potential inhibitor of ATP sulfurylase. The binding mode of compound C revealed favorable interactions with the amino residues LEU 213, ASP 308, ARG 307, TRP 347, LEU 224, GLN 212, MET211 and HIS 309.\n\nImportanceScale formation formed by hydrogen sulfide, which is produced by sulfate reducing bacteria such as Desulfobacter postgatei has been a persistent problem in the oil and gas industry leading to loss of money, time and even lives. The three selected hits from the virtual screenings of about 40 million compounds would possibly inhibit the enzyme, ATP sulfurylase, which is involved in the first reaction in hydrogen sulfide formation in Desulfobacter postgatei. The selected inhibitors are expected to significantly reduce the formation of hydrogen sulfide and consequently prevent the development of pyrite scale in oil and gas wells.

biochemistry↗

bioRxiv2018

Haplopappus platylepis (Asteraceae) resin: an adhesive trap for pest control of crawling arthropods, with antimicrobial potentia

The use of plant secondary metabolites has been incorporated as key part of integrated pest management and as an alternative to the use of pesticides. This may even be more relevant regarding domiciliary pest insects, capable of vectoring pathogens to humans. In these environments control its more difficult due to its possible effect on non-target organisms and human health. Here we evaluated the use of the resinous exudate of Chiles endemic bush Haplopappus platylepis (Asteraceae) as a sticky trap for crawling pest insects. We used Blatta orientalis Linneus (oriental cockroach), a cosmopolitan synanthropic pest, as test organism. We compared effectiveness on cockroach-trapping of H. platylepis resin versus a commercially available sticky trap, and analyzed these two sticky substances using UHPLC-DAD-MS and GC-MS. We found that H. platylepis resin was as effective as the commercial adhesive on trapping B. orientalis. Plant resinous exudate was composed by a mixture of flavonoids, labdane diterpenoids and unsatured fatty acids oxylipins, which are known for their antimicrobial and antioxidant properties. In contrast, the commercial sticky trap was rich in 1-bromohexadecane and 2-clorociclohexanol, which have been described as allergens and as potentially toxic to humans. Considering these findings, we suggest the use of the resinous extract of H. platylepis as an effective adhesive trapping method against pest cockroaches and possibly other crawling synanthropic arthropods cohabiting with humans. We highlight the importance of novel, non-toxic and eco-friendly products as strategies to be applied in the management of insect pests.

biochemistry↗

bioRxiv2018

Minimizing marine ingredients in diets of farmed Atlantic salmon (Salmo salar): Effects on growth performance and muscle lipid and fatty acid composition

Due to limited fish meal and fish oil resources and their high costs for the aquaculture industry, it is necessary to find alternative sustainable sources of protein and lipids. Therefore, seven different diets were formulated with different protein and lipid sources to feed farmed Atlantic salmon, and their effects on growth performance, muscle lipid class, and fatty acid composition were examined. Growth performance indicated that the diet with the lowest fish meal and fish oil content resulted in the lowest weight gain and final weight, followed by the diet containing the highest level of animal by-products. The lipid class analysis showed no statistical difference in the muscle total lipid content using different diets. However, significant statistical differences were observed among the main lipid classes; triacylglycerols, phospholipids, and sterols. The diet containing 1.4% omega-3 long-chain fatty acids resulted in the highest content of triacylglycerols and phospholipids. Diets containing medium and low levels of fish oil and fish meal, respectively, led to as high a level of {omega}3 fatty acids in muscle as when fish were fed diets with high levels of fish meal and fish oil. The results of this study suggest that feeding a diet containing low levels of fish meal and moderate levels of fish oil does not significantly affect {omega}3 fatty acid composition in muscle. Fish meal could be reduced to 5% without affecting growth as long as there was a minimum of 5% fish oil, and animal by-products did not exceed 26% of the diet.

biochemistry↗

bioRxiv2018

Synthesis, Biological Activity and Molecular Docking of New Tricyclic Series as α-glucosidase Inhibitors

Diabetes is an emerging metabolic disorder. -Glucosidase inhibitors, such as acarbose, delay the hydrolysis of carbohydrates by interfering with the digestive enzymes. This action decreases the glucose absorption and the postprandial glucose level. We have synthesized 25 tricyclic 2-phenoxypyrido[3,2-e][1,2,4]triazolo[1,5-a]pyrimidin-5(4H)-ones hybrids and evaluated their -glucosidase inhibitory activity. Compounds 6h and 6d have shown stronger activity than that of acarbose. Compound 6h exhibited the highest inhibition with an IC50 of 104.07 mM. Molecular modelling studies revealed that compound 6h inhibits -glucosidase due to the formation of a stable ligand--glucosidase complex and extra hydrogen bond interactions, and directed in the binding site by Trp329.

biochemistry↗

bioRxiv2018

Luciferase of the Japanese syllid polychaete Odontosyllis umdecimdonta

1Odontosyllis undecimdonta is a marine syllid polychaete that produces bright internal and exuded bioluminescence. Despite over fifty years of biochemical investigation into Odontosyllis bioluminescence, the light-emitting small molecule substrate and catalyzing luciferase protein have remained a mystery. Here we describe the discovery of a bioluminescent protein fraction from O. undecimdonta, the identification of the luciferase using peptide and RNA sequencing, and the in vitro reconstruction of the bioluminescence reaction using highly purified O. undecimdonta luciferin and recombinant luciferase. Lastly, we found no identifiably homologous proteins in publicly available datasets. This suggests that the syllid polychaetes contain an evolutionarily unique luciferase among all characterized luminous taxa.\n\n3 HighlightsO_LIThe polychaete O. undecimdonta uses a luciferin-luciferase bioluminescence system\nC_LIO_LIO. undecimdonta bioluminescence does not require additional cofactors\nC_LIO_LIThe luciferase of the Japanese fireworm is 329 amino acids long\nC_LIO_LIRecombinant luciferase is not secreted when expressed in human cells\nC_LIO_LIExogenous luciferin does not seem to penetrate cell membranes-only lysate luminesces\nC_LIO_LIThe luciferase transcript is supported by full-length cDNA reads with 5 and 3 UTR\nC_LI\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC=\"FIGDIR/small/329631_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (25K):\norg.highwire.dtl.DTLVardef@15882beorg.highwire.dtl.DTLVardef@1efc214org.highwire.dtl.DTLVardef@1aee561org.highwire.dtl.DTLVardef@15582b7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2018

Cell-associated, Heparin-like Molecules Modulate the Ability of LDL to Regulate PCSK9 Uptake

Proprotein convertase subtilisin/kexin type 9 (PCSK9) targets the LDL receptor (LDLR) for degradation, increasing plasma LDL and, consequently, cardiovascular risk. Uptake of secreted PCSK9 is required for its predominant effect on the LDLR. LDL itself inhibits this uptake, though the mechanism by which it does so remains unclear. In this study, we investigated the relationship between LDL, the PCSK9:LDLR interaction, and PCSK9 uptake. We show that LDL inhibits binding of PCSK9 to the epidermal growth factor precursor homology domain A (EGF-A) domain of the LDLR in vitro more impressively than it inhibits PCSK9 uptake in cells. Furthermore, a cell-based factor responsive to heparin-targeting treatments can explain this difference, consistent with its identity as a cell surface heparan sulfate proteoglycan (HSPG), a known co-receptor for PCSK9. Furthermore, we show that the entire PCSK9 prodomain, but not truncated variants, rescues PCSK9 uptake in the presence of LDL, suggesting that PCSK9:LDL binding requires the entire prodomain. Additionally, we show that the gain-of-function (GOF) PCSK9 variant S127R has increased affinity for heparin-like molecules such as HSPGs, potentially explaining the biochemical basis for its phenotype. Overall, our findings suggest a model where PCSK9, LDL, and HSPGs all interact to regulate PCSK9 uptake into the hepatocyte.

biochemistry↗

bioRxiv2018

Towards measuring growth rates of pathogens during infections by D2O-labeling lipidomics

RATIONALEMicrobial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by D2O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids.\n\nMETHODSLipid extracts of D2O-treated E. coli cultures were measured on LC-ESI-MS instruments equipped with TOF and Orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio GC-MS. We then develop an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we apply D2O-labeling lipidomics to clinical samples from CF patients with chronic lung infections.\n\nRESULTSLipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum, indicates an average generation time of the pathogen on the order of one cell doubling per day.\n\nCONCLUSIONSThis study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2O.

biochemistry↗