Biosynthetic oligosaccharide libraries for identification of protein-binding heparan sulfate motifs. Exploring the structural diversity by screening for fibroblast growth factor (FGF)1 and FGF2 binding.
Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various proteins. The identification of protein-binding heparan sulfate sequences is essential to our understanding of heparan sulfate biology and raises the possibility to develop drugs against diseases such as cancer and inflammatory conditions. We present proof-of-principle that in vitro generated heparan sulfate oligosaccharide libraries can be used to explore interactions between heparan sulfate and proteins, and that the libraries expand the available heparan sulfate sequence space. Oligosaccharide libraries mimicking highly 6-O-sulfated domains of heparan sulfate were constructed by enzymatic O-sulfation of O-desulfated, end-group (3)H-labeled heparin octasaccharides. Acceptor oligosaccharides that were 6-O-desulfated but only partially 2-O-desulfated yielded oligosaccharide arrays with increased ratio of iduronyl 2-O-sulfate/glucosaminyl 6-O-sulfate. The products were probed by affinity chromatography on immobilized growth factors, fibroblast growth factor-1 (FGF1) and FGF2, followed by sequence analysis of trapped oligosaccharides. An N-sulfated octasaccharide, devoid of 2-O-sulfate but with three 6-O-sulfate groups, was unexpectedly found to bind FGF1 as well as FGF2 at physiological ionic strength. However, a single 2-O-sulfate group in the absence of 6-O-sulfation gave higher affinity for FGF2. FGF1 binding was also augmented by 2-O-sulfation, preferentially in combination with an adjacent upstream 6-O-sulfate group. These results demonstrate the potential of the enzymatically generated oligosaccharide libraries. (+info)
The biosynthetic pathway for lipoic acid is present in plastids and mitochondria in Arabidopsis thaliana.
In eukaryotes, the biosynthetic pathway for lipoic acid is present in mitochondria. However, it has been hypothesized that, in plants, the biosynthetic pathway is present in plastids in addition to mitochondria. In this study, Arabidopsis thaliana LIP1p cDNA for a plastidial form of lipoic acid synthase has been identified. We show that it encodes a lipoic acid synthase by demonstrating its ability to complement an Escherichia coli mutant lacking lipoic acid synthase activity. We also show that LIP1p is targeted to chloroplasts. These findings suggest that the biosynthetic pathway for lipoic acid is present not only in mitochondria but also in plastids. (+info)
Inhibition of rat liver rhodanese by di-, tricarboxylic, and alpha-keto acids.
Rat liver rhodanese [EC 126.96.36.199] purified by ammonium sulfate fractionation, CM-cellulose and Sephadex G-200 chromatography yielded two active fractions (I & II). Their molecular weights were estimated to be 1.75 X 10(4) (I) and 1.26 X 10(4) (II) by the gel filtration method. Kinetic studies revealed that Fraction I rat liver rhodanese catalyzes thiocyanate formation from thiosulfate and cyanide by a double displacement mechanism. Carboxylic acids such as DL-isocitric, citric malic, pyruvic, and oxaloacetic acid were competitive inhibitors with respect to thiosulfate, whereas fumaric, succinic, and alpha-ketoglutaric acids were noncompetitive inhibitors with respect ot thiosulfate. Incubation of mitochondria with sulfate and alpha-ketoglutaric acid caused a significant decrease in rhodanese activity. (+info)
Evidence from Mossbauer spectroscopy for distinct [2Fe-2S](2+) and [4Fe-4S](2+) cluster binding sites in biotin synthase from Escherichia coli.
Biotin synthase is an AdoMet-dependent radical enzyme that catalyzes the insertion of an FeS cluster-derived sulfur atom into dethiobiotin. The dimeric enzyme is purified containing one [2Fe-2S]2+ cluster per monomer, but it is most active when reconstituted with an additional [4Fe-4S]2+ cluster per monomer. Using Mossbauer spectroscopy coupled with differential reconstitution of each cluster with 57Fe, we show that the reconstituted enzyme has approximately 1:1 [2Fe-2S]2+ and [4Fe-4S]2+ clusters and that the [4Fe-4S]2+ cluster is assembled at an alternate site not previously occupied by the [2Fe-2S]2+ cluster. These data suggest that biotin synthase is evolved to simultaneously accommodate two different clusters with unique roles in catalysis. (+info)
Inhibition of Fe-S cluster biosynthesis decreases mitochondrial iron export: evidence that Yfh1p affects Fe-S cluster synthesis.
Decreased expression of Yfh1p in the budding yeast, Saccharomyces cerevisiae, and the orthologous human gene frataxin results in respiratory deficiency and mitochondrial iron accumulation. The absence of Yfh1p decreases mitochondrial iron export. We demonstrate that decreased expression of Nfs1p, the yeast cysteine desulfurase that plays a central role in Fe-S cluster synthesis, also results in mitochondrial iron accumulation due to decreased export of mitochondrial iron. In the absence of Yfh1p, activity of Fe-S-containing enzymes (aconitase, succinate dehydrogenase) is decreased, whereas the activity of a non-Fe-S-containing enzyme (malate dehydrogenase) is unaffected. Aconitase protein was abundant even though the activity of aconitase was decreased in both aerobic and anaerobic conditions. These results demonstrate a direct role of Yfh1p in the formation of Fe-S clusters and indicate that mitochondrial iron export requires Fe-S cluster biosynthesis. (+info)
Selenium binding to beef-kidney rhodanese.
The reaction of beef kidney rhodanese with selenosulfate was studied. The selenium-treated enzyme shows an absorption spectrum with a maximum at 375 nm attributable to a sulfoselenide group. This absorption is bleached by addition of cyanide. After cyanide treatment stoichiometric amount of selenocyanate can be found. The intrinsic fluorescence of rhodanese is quenched by addition of stoichiometric selenosulfate. This effect can be reversed by cyanide or sulfite but not by selenite or glutathione. By comparison with model complexes the selenium-rhodanese intermediate was identified as a cysteinyl-selenium derivative. (+info)
Inactivation of rhodanese by pyridoxal 5'-phosphate.
Pyridoxal 5'-phosphate and other aromatic aldehydes inactivate rhodanese. The inactivation reaches higher extents if the enzyme is in the sulfur-free form. The identification of the reactive residue as an amino group has been made by spectrophotometric determination of the 5'-phosphorylated pyridoxyl derivative of the enzyme. The inactivation increases with pyridoxal 5'-phosphate concentration and can be partially removed by adding thiosulfate or valine. Prolonged dialysis against phosphate buffer also leads to the enzyme reactivation. The absorption spectra of the pyridoxal phosphate - rhodanese complex show a peak at 410 nm related to the Schiff base and a shoulder in the 330 nm region which is probably due to the reaction between pyridoxal 5'-phosphate and both the amino and thiol groups of the enzyme that appear reasonably close to each other. The relationship betweenloss of activity and pyridoxal 5'-phosphate binding to the enzyme shows that complete inactivation is achieved when four lysyl residues are linked to pyridoxal 5'-phosphate. (+info)
Recombinant Escherichia coli biotin synthase is a [2Fe-2S](2+) protein in whole cells.
EPR and Mossbauer spectroscopies have been used to determine the type and properties of the iron-sulfur clusters present in homologously expressed recombinant Escherichia coli BioB in whole cells prior to purification. Difference EPR spectra of samples of whole cells from a strain over-expressing E. coli BioB and a strain containing the same plasmid but without the bioB insertion showed an axial S=1/2 resonance that was attributed to the [2Fe-2S](+) cluster of the E. coli iron-sulfur cluster assembly 2Fe ferredoxin, based on principal g-values, linewidths and relaxation behavior. Comparison of the Mossbauer spectra of whole cells with and without the bioB insertion revealed that the E. coli cells with over-expressed BioB contain an additional species that exhibits a spectrum identical to that of the [2Fe-2S](2+) cluster in purified recombinant BioB. The concentration of this [2Fe-2S](2+) species in the whole cell sample was quantified using a Mossbauer standard and found to be approximately 260 microM, which was comparable to the BioB protein concentration estimated for the cell paste. The results demonstrate that the [2Fe-2S](2+) cluster found in purified samples of recombinant BioB is not an artifact of the protein purification procedure, and indicate that recombinant BioB is over-expressed in an inactive form during aerobic growth. (+info)