CPExtract, a Software for the Automated Tracer-Based Pathway Specific Screening of Secondary Metabolites in LC-HRMS Data

The use of stable isotopically labeled tracers is a long-proven way of specifically detecting and tracking derived metabolites through a metabolic network of interest. While recently developed stable isotope assisted methods and associated, supporting data analysis tools have greatly improved untargeted metabolomics approaches, no software tool is currently available that allows to automatically search LC-HRMS chromatograms for completely free user-definable isotopolog patterns expected for the metabolism of labeled tracer substances. Here we present Custom Pattern Extract (CPExtract), a versatile software tool that allows for the first time the high-throughput search for user-defined isotopolog patterns in LC-HRMS data. The patterns can be specified via a set of rules including the presence or absence of certain isotopologs, their relative intensity ratios as well as chromatographic co-elution. Each isotopolog pattern satisfying the respective rules is verified on a MS-scan level and also in the chromatographic domain. The CPExtract algorithm allows the use of both labeled tracer compounds in non-labeled biological samples as well as a reversed tracer approach, employing non-labeled tracer compounds along with globally labeled biological samples. In a proof of concept study we searched for metabolites specifically arising from the malonate pathway of the filamentous fungi Fusarium graminearum and Trichoderma reesei. 1,2,3- 13 C 3 -malonic acid diethyl ester and native malonic acid monomethyl ester were used as tracers. We were able to reliably detect expected fatty acids and known polyketides. In addition, up to 189 and 270 further, unknown metabolites presumably including novel polyketides were detected in the F. graminearum and T. reesei culture samples respectively, all of which exhibited the user-predicted isotopolog patterns originating from the malonate tracer incorporation. The software can be used for every conceivable tracer approach. Furthermore, the rule sets can be easily adapted or extended if necessary. CPExtract is available free of charge for non-commercial use at https://metabolomics-ifa.boku.ac.at/CPExtract.

Cytological and Transcriptomic Analysis Provide Insights into the Formation of Variegated Leaves in Ilex × altaclerensis ‘Belgica Aurea’

Ilex × altaclerensis ‘Belgica Aurea’ is an attractive ornamental plant bearing yellow-green variegated leaves. However, the mechanisms underlying the formation of leaf variegation in this species are still unclear. Here, the juvenile yellow leaves and mature variegated leaves of I. altaclerensis ‘Belgica Aurea’ were compared in terms of leaf structure, pigment content and transcriptomics. The results showed that no obvious differences in histology were noticed between yellow and variegated leaves, however, ruptured thylakoid membranes and altered ultrastructure of chloroplasts were found in yellow leaves (yellow) and yellow sectors of the variegated leaves (variegation). Moreover, the yellow leaves and the yellow sectors of variegated leaves had significantly lower chlorophyll compared to green sectors of the variegated leaves (green). In addition, transcriptomic sequencing identified 1675 differentially expressed genes (DEGs) among the three pairwise comparisons (yellow vs. green, variegation vs. green, yellow vs. variegation). Expression of magnesium-protoporphyrin IX monomethyl ester (MgPME) [oxidative] cyclase, monogalactosyldiacylglycerol (MGDG) synthase and digalactosyldiacylglycerol (DGDG) synthase were decreased in the yellow leaves. Altogether, chlorophyll deficiency might be the main factors driving the formation of leaf variegation in I.altaclerensis ‘Belgica Aurea’.

Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls

During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.

Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin

Chlorophyll is the light-harvesting molecule central to the process of photosynthesis. Chlorophyll is synthesized through 15 enzymatic steps. Most of the reactions have been characterized using recombinant proteins. One exception is the formation of the isocyclic E-ring characteristic of chlorophylls. This reaction is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase encoded by Xantha-l in barley (Hordeum vulgare L.). The Xantha-l gene product (XanL) is a membrane-bound diiron monooxygenase, which requires additional soluble and membrane-bound components for its activity. XanL has so far been impossible to produce as an active recombinant protein for in vitro assays, which is required for deeper biochemical and structural analyses. In the present work, we performed cyclase assays with soluble and membrane-bound fractions of barley etioplasts. Addition of antibodies raised against ferredoxin or ferredoxin-NADPH oxidoreductase (FNR) inhibited assays, strongly suggesting that reducing electrons for the cyclase reaction involves ferredoxin and FNR. We further developed a completely recombinant cyclase assay. Expression of active XanL required co-expression with an additional protein, Ycf54. In vitro cyclase activity was obtained with recombinant XanL in combination with ferredoxin and FNR. Our experiment demonstrates that the cyclase is a ferredoxin-dependent enzyme. Ferredoxin is part of the photosynthetic electron-transport chain, which suggests that the cyclase reaction might be connected to photosynthesis under light conditions.