Plant hydroperoxide-cleaving enzymes (CYP74 family) function as hemiacetal synthases: Structural proof of hemiacetals by NMR spectroscopy

Hydroperoxide lyases (HPLs) of the CYP74 family (P450 superfamily) are widely distributed enzymes in higher plants and are responsible for the stress-initiated accumulation of short-chain aldehydes. Fatty acid hydroperoxides serve as substrates for HPLs; however, details of the HPL-promoted conversion are still incompletely understood. In the present work, we report first time the micropreparative isolation and the NMR structural studies of fatty acid hemiacetal (TMS/TMS), the short-lived HPL product. With this aim, linoleic acid 9(S)?hydroperoxide (9(S)?HPOD) was incubated with recombinant melon hydroperoxide lyase (CmHPL, CYP74C2) in a biphasic system of water/hexane for 60?s at 0?°C, pH?4.0. The hexane layer was immediately decanted and vortexed with a trimethylsilylating mixture. Analysis by GC–MS revealed a major product, i.e. the bis-TMS derivative of a hemiacetal which was conclusively identified as 9?hydroxy?9?[(1′E,3′Z)?nonadienyloxy]?nonanoic acid by NMR-spectroscopy. Further support for the hemiacetal structure was provided by detailed NMR-spectroscopic analysis of the bis-TMS hemiacetal generated from [¹³C₁₈]9(S)?HPOD in the presence of CmHPL. The results obtained provide incontrovertible evidence that the true products of the HPL group of enzymes are hemiacetals, and that the short-chain aldehydes are produced by their rapid secondary chain breakdown. Therefore, we suggest replacing the name “hydroperoxide lyase”, which does not reflect the factual isomerase (intramolecular oxidoreductase) activity, with “hemiacetal synthase” (HAS).     

Assessment of magnetic fields surrounding electronic article surveillance systems in Sweden

Magnetic fields surrounding 11 EAS systems in Swedish shops and libraries were measured, in real-life conditions, according to the CENELEC standard. For each system, the arithmetic mean of 45 measurement points was compared to ICNIRP's reference level. Six of eleven measured systems exceeded the reference levels.     

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Showing a Covalent Flavin-Aspartate Bond

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH’s). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 Å resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique ‘winged-helix’ C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4α)-OOH intermediate. Strikingly, the 8α carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.     

Design and coverage of high throughput genotyping arrays optimized for individuals of East Asian, African American, and Latino race/ethnicity using imputation and a novel hybrid SNP selection algorithm

Four custom Axiom genotyping arrays were designed for a genome-wide association (GWA) study of 100,000 participants from the Kaiser Permanente Research Program on Genes, Environment and Health. The array optimized for individuals of European race/ethnicity was previously described. Here we detail the development of three additional microarrays optimized for individuals of East Asian, African American, and Latino race/ethnicity. For these arrays, we decreased redundancy of high-performing SNPs to increase SNP capacity. The East Asian array was designed using greedy pairwise SNP selection. However, removing SNPs from the target set based on imputation coverage is more efficient than pairwise tagging. Therefore, we developed a novel hybrid SNP selection method for the African American and Latino arrays utilizing rounds of greedy pairwise SNP selection, followed by removal from the target set of SNPs covered by imputation. The arrays provide excellent genome-wide coverage and are valuable additions for large-scale GWA studies.     

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the “hydroperoxide-binding domain” (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs.     

Polyunsaturated C18 fatty acids derivatized with Gly and Ile as an additional tool for studies of the catalytic evolution of fungal 8- and 9-dioxygenases

The fungal linoleate diol synthase (LDS) family contains over twenty characterized 8-, 9-, and 10-dioxygenases (DOX), usually fused to catalytically competent cytochromes P450. Crystal structures are not available, but indirect evidence suggests that linoleic acid enters the active site of 8R-DOX-LDS headfirst and enters 9S-DOX-allene oxide synthase (AOS) with the ω-end (tail) first. Fatty acids derivatized with amino acids can conceivably be used to study oxidation in tail first position by enzymes, which bind natural fatty acids headfirst. The results might reveal catalytic similarities of homologous enzymes. 8R-DOX-5,8-LDS oxidize 18:2n-6-Ile and 18:2n-6-Gly in tail first position to 9S-hydroperoxy metabolites, albeit with less position and stereo specificity than 9S-DOX-AOS. The oxygenation mechanism of 9S-DOX-AOS with antarafacial hydrogen abstraction at C-11 and oxygen insertion at C-9 was also retained. Two homologues, 8R-DOX-7,8-LDS and 8R-DOX-AOS, oxidized 18:2n-6-Ile and 18:2n-6-Gly at C-9, suggesting a conserved feature of 8R-DOX domains. 9R-DOX-AOS, with 54% sequence identity to 9S-DOX-AOS, did not oxidize the derivatized C₁₈ fatty acids. 9Z,12Z-16:2, two carbon shorter than 18:n-6 from the ω-end, was rapidly metabolized to an α-ketol, but 7Z,10Z-16:2 was not a substrate. An unsaturated carbon chain from C-1 to C-8 was apparently more important than the configuration at the ω-end. 8R-DOX-LDS and 9R-DOX-AOS may thus bind 18:2n-6 in the same orientation. The oxidation of 18:2n-6 in straight or reverse head-to-tail positions illustrates evolutionary traits between 8- and 9-DOX domains. Fatty acids derivatized with amino acids provide a complementary tool for the analysis of evolution of enzymes.