Proline hydroxylases are iron(II)/2-oxoglutarate-dependent enzymes that hydroxylate l-proline and derivatives, such as lpipecolic acid, which is the six-membered-ring homologue of l-proline. It has been established that there is a distinct group of conserved bacterial enzymes that hydroxylate l-pipecolic acid and trans-3- and trans-4-methyl-l-proline, but virtually no l-proline. This allows the organism to produce hydroxyproline congeners without hydroxylation of the physiologically omnipresent l-proline. In vitro conversions showed that the substrate spectrum of the pipecolic acid hydroxylases GetF (from a Streptomyces sp.; producer of the tetrapeptide antibiotic GE81112) and PiFa (from Frankia alni) overlaps that of proline hydroxylases, except for the nonacceptance of l-proline and smaller homologues. Distinct and conserved residues were determined for both types of enzymes. However, site-directed mutagenesis in GetF did not yield variants that accepted l-proline; this suggested a complex interaction of several residues around the active site, which resulted in delicate changes in substrate specificity. This is supported by substrate docking in a homology model of GetF, which revealed an altered orientation for l-proline relative to that of preferred substrates.
Echinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines: -4-hydroxy-l-proline and, in most echinocandin structures, (-2,3)-3-hydroxy-(-2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis by , both amino acids are found in pneumocandin A, while in pneumocandin B the latter residue is replaced by -3-hydroxy-l-proline (3-Hyp). We have recently reported that all three amino acids are generated by the 2-oxoglutarate-dependent proline hydroxylase GloF. In echinocandin B biosynthesis by species, 3-Hyp derivatives have not been reported. Here we describe the heterologous production and kinetic characterization of HtyE, the 2-oxoglutarate-dependent proline hydroxylase from the echinocandin B biosynthetic cluster in Surprisingly, l-proline hydroxylation with HtyE resulted in an even higher proportion (∼30%) of 3-Hyp than that with GloF. This suggests that the selectivity for methylated 3-Hyp in echinocandin B biosynthesis is due solely to a substrate-specific adenylation domain of the nonribosomal peptide synthetase. Moreover, we observed that one product of HtyE catalysis, 3-hydroxy-4-methyl-l-proline, is slowly further oxidized at the methyl group, giving 3-hydroxy-4-hydroxymethyl-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced by Secondary metabolites from bacteria and fungi are often produced by sets of biosynthetic enzymes encoded in distinct gene clusters. Usually, each enzyme catalyzes one biosynthetic step, but multiple reactions are also possible. Pneumocandins A and B are produced by the fungus They belong to the echinocandin family, a group of nonribosomal cyclic lipopeptides that exhibit a strong antifungal activity. Chemical derivatives are important drugs for the treatment of systemic fungal infections. We have recently shown that in the biosynthesis of pneumocandins A and B, three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis in produces the same hydroxyprolines, with an increased proportion of-3-hydroxyproline. However, echinocandin B biosynthesis does not require -3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation in and provides an unusual insight into peptide antibiotic biosynthesis-namely, the complex interplay between the selectivity of a hydroxylase and the substrate specificity of a nonribosomal peptide synthetase.
The 5 0 cap of eukaryotic mRNA is a hallmark for cellular functions from mRNA stability to translation. However, the discovery of novel 5 0 -terminal RNA caps derived from cellular metabolites has challenged this long-standing singularity in both eukaryotes and prokaryotes. Reminiscent of the 7-methylguanosine (m7G) cap structure, these noncanonical caps originate from abundant coenzymes such as NAD, FAD, or CoA and from metabolites like dinucleoside polyphosphates (NpnN). As of now, the significance of noncanonical RNA caps is elusive: they differ for individual transcripts, occur in distinct types of RNA, and change in response to environmental stimuli. A thorough comparison of their prevalence, quantity, and characteristics is indispensable to define the distinct classes of metabolite-capped RNAs. This is achieved by a structured analysis of all present studies covering functional, quantitative, and sequencing data which help to uncover their biological impact. The biosynthetic strategies of noncanonical RNA capping and the elaborate decapping machinery reveal the regulation and turnover of metabolite-capped RNAs. With noncanonical capping being a universal and ancient phenomenon, organisms have developed diverging strategies to adapt metabolite-derived caps to their metabolic needs, but ultimately to establish noncanonical RNA caps as another intriguing layer of RNA regulation.
It is commonly understood that RNA-binding proteins crucially determine the fate of their target RNAs. Vice versa, RNAs are becoming increasingly recognized for their functions in protein regulation and the dynamics of RNA-protein complexes. Long non-coding RNAs are emerging as potent regulators of proteins that exert unknown RNA-binding properties and moonlighting functions. A vast array of RNA- and protein-centric techniques have been developed for the identification of protein and RNA targets, respectively, including unbiased protein mass spectrometry and next-generation RNA sequencing as readout. Determining true physiological RNA and protein targets is challenging as RNA–protein interaction is highly dynamic, tissue- and cell-type-specific, and changes with the environment. Here I review current techniques for the analysis of RNA–protein interactions in living cells and in vitro. RNA-centric techniques are presented on the basis of cross-linking or the use of alternative approaches. Protein-centric approaches are discussed in combination with high-throughput sequencing. Finally, the impact of mutations in RNA–protein complexes on human disease is highlighted.
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