Same but different — pseudo-pectin in the charophytic alga Chlorokybus atmophyticus

All land‐plant cell walls possess hemicelluloses, cellulose and anionic pectin. The walls of their cousins, the charophytic algae, exhibit some similarities to land plants’ but also major differences. Charophyte ‘pectins’ are extractable by conventional land‐plant methods, although they differ significantly in composition. Here, we explore ‘pectins’ of an early‐diverging charophyte, Chlorokybus atmophyticus, characterising the anionic polysaccharides that may be comparable to ‘pectins’ in other streptophytes. Chlorokybus ‘pectin’ was anionic and upon acid hydrolysis gave GlcA, GalA and sulphate, plus neutral sugars (Ara≈Glc>Gal>Xyl); Rha was undetectable. Most Gal was the l‐enantiomer. A relatively acid‐resistant disaccharide was characterised as β‐d‐GlcA‐(1→4)‐l‐Gal. Two Chlorokybus ‘pectin’ fractions, separable by anion‐exchange chromatography, had similar sugar compositions but different sulphate‐ester contents. No sugars were released from Chlorokybus ‘pectin’ by several endo‐hydrolases [(1,5)‐α‐l‐arabinanase, (1,4)‐β‐d‐galactanase, (1,4)‐β‐d‐xylanase, endo‐polygalacturonase] and exo‐hydrolases [α‐ and β‐d‐galactosidases, α‐(1,6)‐d‐xylosidase]. ‘Driselase’, which hydrolyses most land‐plant cell wall polysaccharides to mono‐ and disaccharides, released no sugars except traces of starch‐derived Glc. Thus, the Ara, Gal, Xyl and GalA of Chlorokybus ‘pectin’ were not non‐reducing termini with configurations familiar from land‐plant polysaccharides (α‐l‐Araf, α‐ and β‐d‐Galp, α‐ and β‐d‐Xylp and α‐d‐GalpA), nor mid‐chain residues of α‐(1→5)‐l‐arabinan, β‐(1→4)‐d‐galactan, β‐(1→4)‐d‐xylan or α‐(1→4)‐d‐galacturonan. In conclusion, Chlorokybus possesses anionic ‘pectic’ polysaccharides, possibly fulfilling pectic roles but differing fundamentally from land‐plant pectin. Thus, the evolution of land‐plant pectin since the last common ancestor of Chlorokybus and land plants is a long and meandering path involving loss of sulphate, most l‐Gal and most d‐GlcA; re‐configuration of Ara, Xyl and GalA; and gain of Rha

Development And Application Of Chemical Tools For The Study Of S-Adenosyl-L-Methionine-Dependent Methyltransferases

Methyltransferases represent a class of enzyme responsible for the modification of biomolecules through the transfer of individual methyl units. The cofactor, S-adenosyl-L-methionine (SAM), serves as the methyl source for the vast majority of these enzyme-catalyzed reactions. These transformations have broad implications for many biological processes, ranging from the biosynthesis of essential cellular metabolites and pharmaceutically relevant natural products to the regulation of gene expression and protein function through the modification of nucleic acids and polypeptides. In addition, the malfunction of methyltransferase activity has been strongly implicated in a number of disease states including developmental disorders and carcinogenesis. As such, there has been significant effort in recent years to better understand these enzymes, their substrates, and the biological effects associated with their activity. Despite increased interest, the study of these processes has proven difficult using traditional biochemical or genetic techniques. In light of this, the research described herein has been aimed at the development of novel chemical tools and approaches for the study of these enzymes, with an emphasis on protein methyltransferases (PMTs). This research can be broadly categorized into two main focuses: (i) the implementation of Bioorthogonal Profiling of Protein Methylation (BPPM), in which substrates of specific PMTs are determined through the use of engineered enzymes, SAM analogues and bioorthogonal chemistry; and (ii) the development of a selenium-based SAM analogues, one of which has shown broad compatibility toward a wide variety of wild-type enzymes including: protein, nucleic acid and small-molecule methyltransferases. With these tools in hand, novel substrates for the G9a and GLP1 protein lysine methyltransferases have been identified, and a versatile selenium-based SAM mimic has demonstrated potential as a useful tool for the enzymatic functionalization of proteins and small molecules

Large-Scale, Protection-Free Synthesis of <i>Se</i>-Adenosyl‑l‑selenomethionine Analogues and Their Application as Cofactor Surrogates of Methyltransferases

<i>S</i>-Adenosyl-l-methionine (SAM) analogues have previously demonstrated their utility as chemical reporters of methyltransferases. Here we describe the facile, large-scale synthesis of <i>Se</i>-alkyl <i>Se</i>-adenosyl-l-selenomethionine (SeAM) analogues and their precursor, <i>Se</i>-adenosyl-l-selenohomocysteine (SeAH). Comparison of SeAM analogues with their equivalent SAM analogues suggests that sulfonium-to-selenonium substitution can enhance their compatibility with certain protein methyltransferases, favoring otherwise less reactive SAM analogues. Ready access to SeAH therefore enables further application of SeAM analogues as chemical reporters of diverse methyltransferases

The properties of the star-forming interstellar medium at z=0.8-2.2 from HiZELS II : star formation and clump scaling laws in gas-rich, turbulent disks

We present adaptive optics assisted integral field spectroscopy of nine Ha-selected galaxies at z = 0.84-2.23 drawn from the HiZELS narrowband survey. Our observations map the kinematics of these star-forming galaxies on similar to kpc scales. We demonstrate that within the interstellar medium of these galaxies, the velocity dispersion of the star-forming gas (sigma) follows a scaling relation sigma alpha Sigma(1/n)(SFR) constant (where Sigma(SFR) is the star formation surface density and the constant includes the stellar surface density). Assuming the disks are marginally stable (Toomre Q = 1), this follows from the Kennicutt-Schmidt relation (Sigma(SFR) = A Sigma(n)(gas)), and we derive best-fit parameters of n = 1.34 +/- 0.15 and A = 3.4(-1.6)(+2.5) x 10(-4) M-circle dot yr(-1) kpc(-2), consistent with the local relation, and implying cold molecular gas masses of M-gas = 10(9-10) M-circle dot and molecular gas fractions of M-gas/(M-gas + M-star) = 0.3 +/- 0.1, with a range of 10%-75%. We also identify 11 similar to kpc-scale star-forming regions (clumps) within our sample and show that their sizes are comparable to the wavelength of the fastest growing mode. The luminosities and velocity dispersions of these clumps follow the same scaling relations as local H II regions, although their star formation densities are a factor similar to 15 +/- 5 x higher than typically found locally. We discuss how the clump properties are related to the disk, and show that their high masses and luminosities are a consequence of the high disk surface density.Peer reviewe

Expanding the Structural Diversity of Polyketides by Exploring the Cofactor Tolerance of an Inline Methyltransferase Domain

A strategy for introducing structural diversity into polyketides by exploiting the promiscuity of an in-line methyltransferase domain in a multidomain polyketide synthase is reported. In vitro investigations using the highly-reducing fungal polyketide synthase CazF revealed that its methyltransferase domain accepts the nonnatural cofactor propargylic <i>Se</i>-adenosyl-l-methionine and can transfer the propargyl moiety onto its growing polyketide chain. This propargylated polyketide product can then be further chain-extended and cyclized to form propargyl-α pyrone or be processed fully into the alkyne-containing 4′-propargyl-chaetoviridin A

Bioorthogonal Profiling of Protein Methylation Using Azido Derivative of <i>S</i>-Adenosyl-l-methionine

Protein methyltransferases (PMTs) play critical roles in multiple biological processes. Because PMTs often function in vivo through forming multimeric protein complexes, dissecting their activities in the native contexts is challenging but relevant. To address such a need, we envisioned a Bioorthogonal Profiling of Protein Methylation (BPPM) technology, in which a SAM analogue cofactor can be utilized by multiple rationally engineered PMTs to label substrates of the corresponding native PMTs. Here, 4-azidobut-2-enyl derivative of <i>S</i>-adenosyl-l-methionine (Ab-SAM) was reported as a suitable BPPM cofactor. The resultant cofactor–enzyme pairs were implemented to label specifically the substrates of closely related PMTs (e.g., EuHMT1 and EuHMT2) in a complex cellular mixture. The BPPM approach, coupled with mass spectrometric analysis, enables the identification of the nonhistone targets of EuHMT1/2. Comparison of EuHMT1/2’s methylomes indicates that the two human PMTs, although similar in terms of their primary sequences, can act on the distinct sets of nonhistone targets. Given the conserved active sites of PMTs, Ab-SAM and its use in BPPM are expected to be transferable to other PMTs for target identification

<i>Se</i>-Adenosyl‑l‑selenomethionine Cofactor Analogue as a Reporter of Protein Methylation

Posttranslational methylation by <i>S</i>-adenosyl-l-methionine­(SAM)-dependent methyltransferases plays essential roles in modulating protein function in both normal and disease states. As such, there is a growing need to develop chemical reporters to examine the physiological and pathological roles of protein methyltransferases. Several sterically bulky SAM analogues have previously been used to label substrates of specific protein methyltransferases. However, broad application of these compounds has been limited by their general incompatibility with native enzymes. Here we report a SAM surrogate, ProSeAM (propargylic <i>Se</i>-adenosyl-l-selenomethionine), as a reporter of methyltransferases. ProSeAM can be processed by multiple protein methyltransferases for substrate labeling. In contrast, sulfur-based propargylic SAM undergoes rapid decomposition at physiological pH, likely via an allene intermediate. In conjunction with fluorescent/affinity-based azide probes, copper-catalyzed azide–alkyne cycloaddition chemistry, in-gel fluorescence visualization and proteomic analysis, we further demonstrated ProSeAM’s utility to profile substrates of endogenous methyltransferases in diverse cellular contexts. These results thus feature ProSeAM as a convenient probe to study the activities of endogenous protein methyltransferases