Using S-adenosyl-l-homocysteine capture compounds to characterize S-adenosyl-l-methionine and S-adenosyl-l-homocysteine binding proteins

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH–CC with biotin used in conjunction with streptavidin–horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the K_d values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established K_d values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.     

Enzymatic stereospecific preparation of fluorescent S-adenosyl-l-methionine analogs

S-Adenosyl-l-methionine (SAM) is the preferred cofactor for biological methyl group transfers to various substrates such as nucleic acids, proteins, and lipids. Here we present stereospecific (>95% of the desired enantiomer) and high-yield preparation of four fluorescent and biologically active SAM analogs and demonstrate their usefulness in binding studies. Using a fluorescence titration experiment, we obtained a K_d of 0.38 μM for the S-2,6-diaminopurinylmethionine-SAM-III riboswitch complex.     

A fast and efficient method for quantitative measurement of S-adenosyl-l-methionine-dependent methyltransferase activity with protein substrates

Modification of protein residues by S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis-Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1 h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.     

Structural characterization reveals that viperin is a radical S-adenosyl-l-methionine (SAM) enzyme

Viperin is an interferon-inducible protein inhibiting many DNA and RNA viruses. It contains an N-terminal transmembrane helix, a highly conserved C-terminus and a middle region carrying a CX3CX2C motif, characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. So far no structural characterization has been reported and reconstitution of the [4Fe-4S] cluster in viperin all failed. Here, by dissecting the 361-residue human viperin into 12 fragments, followed by extensive CD and NMR characterization, Viperin (45-361) was identified to be soluble and structured in buffers. Most importantly, we have successfully reconstituted the [4Fe-4S] cluster in Viperin (45-361), thus providing the first experimental evidence confirming that viperin is indeed a radical SAM enzyme. Furthermore, the C-terminus Viperin (214-361) which is insoluble in buffers but again can be solubilized in salt-free water appears to be only partially folded. Our results thus imply that the radical SAM enzyme activity may play a key role in the broad antiviral actions of viperin.     

The enzymatic synthesis of S-adenosyl-l-[2(n)-H]homocysteine

A rapid, efficient method is described for the enzymatic conversion of S-adenosyl-l-[2(n)-³H]methionine to S-adenosyl-l-[2(n)-³H]homocysteine. Partially purified glycine N-methyltransferase is used in the reaction which yields 98% conversion. The product is purified using high-pressure liquid chromatography and is concentrated by lyophilization. S-Adenosyl-l-[2(n)-³H]homocysteine synthesized by this method is an active substrate for S-adenosylhomocysteine (SAH) hydrolase. A novel assay procedure for SAH hydrolase is also described, in which unreacted S-adenosyl-l-[2(n)-³H]homocysteine is removed by adsorption to dextran-coated charcoal.     

Structure of N-acetyl-l-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor l-arginine bound

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in l-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by l-arginine, although l-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by l-arginine, we have determined the structure of the mmNAGS/K complexed with l-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of l-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the l-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when l-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by l-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism.     

Single-step bioconversion for the preparation of l-gulose and l-galactose

Both carbohydrate monomers l-gulose and l-galactose are rarely found in nature, but are of great importance in pharmacy R&D and manufacturing. A method for the production of l-gulose and l-galactose is described that utilizes recombinant Escherichia coli harboring a unique mannitol dehydrogenase. The recombinant E. coli system was optimized by genetic manipulation and directed evolution of the recombinant protein to improve conversion. The resulting production process requires a single step, represents the first readily scalable system for the production of these sugars, is environmentally friendly, and utilizes inexpensive reagents, while producing l-galactose at 4.6 g L⁻¹ d⁻¹ and l-gulose at 0.90 g L⁻¹ d⁻¹.     

Structural Basis for Catalysis by the Mono- and Dimetalated Forms of the dapE-Encoded N-succinyl-l,l-Diaminopimelic Acid Desuccinylase

Biosynthesis of lysine and meso-diaminopimelic acid in bacteria provides essential components for protein synthesis and construction of the bacterial peptidoglycan cell wall. The dapE operon enzymes synthesize both meso-diaminopimelic acid and lysine and, therefore, represent potential targets for novel antibacterials. The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase functions in a late step of the pathway and converts N-succinyl-l,l-diaminopimelic acid to l,l-diaminopimelic acid and succinate. Deletion of the dapE gene is lethal to Helicobacter pylori and Mycobacterium smegmatis, indicating that DapE's are essential for cell growth and proliferation. Since there are no similar pathways in humans, inhibitors that target DapE may have selective toxicity against only bacteria. A major limitation in developing antimicrobial agents that target DapE has been the lack of structural information. Herein, we report the high-resolution X-ray crystal structures of the DapE from Haemophilus influenzae with one and two zinc ions bound in the active site, respectively. These two forms show different activity. Based on these newly determined structures, we propose a revised catalytic mechanism of peptide bond cleavage by DapE enzymes. These structures provide important insight into catalytic mechanism of DapE enzymes as well as a structural foundation that is critical for the rational design of DapE inhibitors.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

d-Polyglutamine Amyloid Recruits l-Polyglutamine Monomers and Kills Cells

Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either l- or d-polyQ peptides and found that d-fibrils are as cytotoxic as l-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced l-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized d- and l-polyQ in vitro. We found that, as expected, d-polyQ monomers are not recognized by proteins that recognize l-polyQ monomers. However, amyloid fibrils prepared from d-polyQ peptides can efficiently seed the aggregation of l-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a “rippled β-sheet” interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.     

Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing l-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-l-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-l-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel l-proline cis-4-hydroxylases converting free l-proline to cis-4-hydroxy-l-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected l-proline cis-4-hydroxylase activity in both proteins. Likewise l-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).     

Metabolic studies of rat liver plasma membranes using d-[l-C]glucosamine

The metabolism of plama membranes of rat liver cells was studied using d-[l-¹⁴C]glucosamine. The labelling of plasma membranes occurred more slowly than that of microsomes, reaching a maximum at about 3 h after injection compared to 1.5 h for microsomes, and the radioactivity decayed with a half-life of 37 h which is close to the value obtained using [guanidino-¹⁴C]arginine to label proteins. Hexosamine and sialic acid of plasma membranes were found to metabolize at practically equal rates.     

A novel l-isoleucine hydroxylating enzyme, l-isoleucine dioxygenase from Bacillus thuringiensis, produces (2S,3R,4S)-4-hydroxyisoleucine

The unique function of 4-hydroxyisoleucine (4-HIL) is to stimulate glucose-induced insulin secretion in a glucose-dependent manner. 4-HIL is distributed only in certain kinds of plants and mushrooms, but the biosynthetic mechanism of 4-HIL has not been elucidated. Moreover, 4-HIL-producing microorganisms have not been reported. l-isoleucine (l-Ile) hydroxylating activity producing 4-HIL was detected in a cell lysate of Bacillus thuringiensis strain 2e2 AKU 0251 obtained from the mid-late exponential phase of growth. Properties of the purified hydroxylase demonstrated that it is a α-ketoglutaric acid (α-KG) dependent l-Ile dioxygenase (IDO) and requires α-KG, ferric ion, and ascorbic acid for its maximum activity. IDO showed high stereoselectivity in l-Ile hydroxylation producing only (2S,3R,4S)-4-HIL. The N-terminal 22 amino acids sequence revealed high homology to a hypothetical protein (GenBank ID: RBTH_06809) in B. thuringiensis serovar israelensis ATCC 35646. The histidine motif, which is conserved in α-KG dependent dioxygenases, is found in RBTH_06809.     

Preparation of NG-monoethyl-l-arginine

N^G-Monoethyl-l-arginine, a putative in vivo product after administration of the potent hepatocarcinogen l-ethionine to rats, has been chemically synthesized by coupling N-ethyl, S-methylthiopseudouronium iodide with α-amino-blocked l-ornithine. The structure of the compound as N^G-monoethyl-l-arginine was confirmed by ¹³C NMR. Its elution time on an automatic amino acid analyzer, R_f values using thin-layer chromatography, and isoelectric point have been compared with those of N^G-monomethyl-l-arginine.     

Efficient and practical synthesis of l-hamamelose

An efficient synthetic route of l-hamamelose was successfully accomplished starting from d-ribose. l-Hamamelose was synthesized in 42% overall yield with six reaction steps via a stereoselective Grignard reaction, a stereoselective crossed aldol reaction and a controlled oxidative cleavage of the double bond of a vinyl diol compound. During the oxidative cleavage of the double bond of the vinyl diol compound with osmium tetroxide and NaIO₄, an over-oxidative cleavage of α-hydroxyl aldehyde generated from ring opening of the first cleaved product, formyl lactol, did not occur, probably due to the stability of the lactol form. A plausible mechanism for the stereoselective crossed aldol reaction was suggested. The final target compound, l-hamamelose can play a very important role as a chiral building block in synthesizing a wide variety of enantiopure compounds.     

Structural insights into conserved l-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic l-arabinose isomerase

Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilusl-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of l-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds.     

Homochiral metal-organic coordination networks from l-typtophan

Three homochiral metal-organic coordination networks [Co₂(l-Trp)₂(Py)₆] · Py · (ClO₄)₂ (1), [Ni(l-Trp)(Py)₃] · H₂O · ClO₄ (2) and [Co₂(l-Trp)(INT)₂(H₂O)₂(ClO₄)] (3), all containing natural amino acid l-HTrp (l-typtophan), were hydrothermally synthesized and structurally characterized. The compounds 1 and 2 crystallize in the orthorhombic space group C222₁, with a = 10.731(2) Å, b = 19.709(4) Å, c = 27.365(6) Å and Z = 4 for 1 and a = 10.710(10) Å, b = 20.088(18) Å, c = 27.63(3) Å and Z = 8 for 2, respectively. The compound 3 has the monoclinic space group P2₁, with a = 8.1934(14) Å, b = 13.209(2) Å, c = 12.464(2) Å, β = 104.107(3)° and Z = 2. Both 1 and 2 consist of 1D helical chains. Compound 3 is composed of 2D networks, which further assemble into a 3D supramolecular structure via weak interlayer interactions. The optically pure amino acid l-HTrp plays an important role leading to homochiral structures reported here.     

Cloning and in vitro characterization of dTDP-6-deoxy-l-talose biosynthetic genes from Kitasatospora kifunensis featuring the dTDP-6-deoxy-l-lyxo-4-hexulose reductase that synthesizes dTDP-6-deoxy-l-talose

Kitasatospora kifunensis, the talosin producer, was used as a source for the dTDP-6-deoxy-l-talose (dTDP-6dTal) biosynthetic gene cluster, serving as a template for four recombinant proteins of RmlA_Kkf, RmlB_Kkf, RmlC_Kkf, and Tal, which complete the biosynthesis of dTDP-6dTal from dTTP, α-d-glucose-1-phosphate, and NAD(P)H. The identity of dTDP-6dTal was validated using ¹H and ¹³C NMR spectroscopy. K. kifunensistal and tll, the known dTDP-6dTal synthase gene of Actinobacillus actinomycetemcomitans origin, have low sequence similarity and are distantly related within the NDP-6-deoxy-4-ketohexose reductase family, providing an example of the genetic diversity within the dTDP-6dTal biosynthetic pathway.