Elucidating the cyclization cascades in xiamycin biosynthesis by substrate synthesis and enzyme characterizations.

Indolosesquiterpene xiamycin A features a pentacyclic core structure. The chemical synthesis of two key precursors, 3-farnesylindole and 3-(epoxyfarnesyl)-indole, allowed elucidation of the enzymatic cascades forming the pentacyclic ring system of xiamycin A by XiaO-catalyzed epoxidation and the membrane protein XiaH-catalyzed terpene cyclization. The substrate flexibility of XiaI, an indole oxygenase for assembly of the central ring, was also demonstrated.

Engineering ß-lactamase zymogens for use in protease activity assays.

In this study, the concept of autoinhibition, a mechanism of protein activity regulation, was applied to the design and engineering of a ß-lactamase zymogen. Using this zymogen, a sensitive protease assay method was developed in which activation of the zymogen by proteases produces an amplified absorbance signal. The approach reported here can be adapted for engineering of zymogens as biological sensors and components of synthetic signaling pathways.

The conversion of a phenol to an aniline occurs in the biochemical formation of the 1-(4-aminophenyl)-1-deoxy-D-ribitol moiety in methanopterin.

Recent work has demonstrated that 4-hydroxybenzoic acid is the in vivo precursor to the 1-(4-aminophenyl)-1-deoxy-D-ribitol (APDR) moiety present in the C(1) carrier coenzyme methanopterin present in the methanogenic archaea. For this transformation to occur, the hydroxyl group of the 4-hydroxybenzoic acid must be replaced with an amino group at some point in the biosynthetic pathway. Using stable isotopically labeled precursors and liquid chromatography with electrospray-ionization mass spectroscopy, the first step of this transformation in Methanocaldococcus jannaschii occurs by the reaction of 4-hydroxybenzoic acid with phosphoribosyl pyrophosphate (PRPP) to form 4-(ß-d-ribofuranosyl)hydroxybenzene 5'-phosphate (ß-RAH-P). The ß-RAH-P then condenses with l-aspartate in the presence of ATP to form 4-(ß-d-ribofuranosyl)-N-succinylaminobenzene 5'-phosphate (ß-RFSA-P). Elimination of fumarate from ß-RFSA-P produces 4-(ß-D-ribofuranosyl)aminobenzene 5'-phosphate (ß-RFA-P), the known precursor to the APDR moiety of methanopterin [White, R. H. (1996) Biochemistry 35, 3447-3456]. This work represents the first biochemical example of the conversion of a phenol to an aniline.

Inhibitory activity and structural characterization of a C-terminal peptide fragment derived from the prosegment of the proprotein convertase PC7.

Mammalian proprotein convertases (PCs) belong to the family of recently discovered serine proteases responsible for the processing of a large number of precursor proteins into their active forms. The enzymatic activities of the convertases have been implicated in a variety of disease states, such as cancer and infectious and inflammatory diseases. Like many other proteases, PCs are also synthesized as inactive proenzymes with N-terminal extensions as their prosegments. Here, we present the inhibitory activities of a number of "putative" interfacial peptide fragments derived from the proregion of PC7. We found that a peptide fragment corresponding to the C-terminal region (residues 81p-104p, or C24: E(1)-A-V-L-A-K-H-E-A-V-R-W-H-S-E-Q-R-L-L-K-R-A-K-R(24)) of the PC7 prosegment displays a strong inhibition (K(i) = 7 nM) of the PC7 enzyme comparable to that of the full-length (104 residue) prosegment. The same 24 residue peptide shows significantly populated helical conformations in an aqueous solution close to the physiological condition. Structure calculations driven by NOE distance restraints revealed a slightly kinked helical conformation for the entire peptide, characterized by many side-chain/side-chain interactions including those involving charged residues E8-R11-E15 and hydrophobic residues W12 and L19. These results suggest that the C-terminal region of the prosegment of PC7 may play a dominant role in conferring the inhibitory potency to the cognate enzyme and this strong inhibitory activity may be a direct consequence of the folded conformation of the peptide fragment in solution. We surmise that such a structure-function correlation for an inhibitory peptide could lead to the design and discovery of molecules mimicking the specific interactions of the PC prosegments for their cognate proteases.

Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma.

Granzymes are granule-stored lymphocyte serine proteases that are implicated in T- and natural killer cell-mediated cytotoxic defense reactions after target cell recognition. A fifth human granzyme (granzyme 3, lymphocyte tryptase-2), renamed as granzyme K (gene name GZMK), has recently been cloned from lymphocyte tissue. For its further characterization we successfully generated catalytically active enzyme in milligram quantities per liter of Escherichia coli culture. The natural proform of granzyme K with the amino-terminal propeptide Met-Glu was expressed as inclusion bodies and converted to its active enzyme by cathepsin C after refolding of precursor molecules. Recombinant granzyme K cleaves synthetic thiobenzyl ester substrates after Lys and Arg with k(cat)/K(m) values of 3.7 x 10(4) and 4.4 x 10(4) M(-1) s(-1), respectively. Granzyme K activity was shown to be inhibited by the synthetic compounds Phe-Pro-Arg-chloromethyl ketone, phenylmethylsulfonyl fluoride, PefablocSC, and benzamidine, by the Kunitz-type inhibitor aprotinin and by human blood plasma. The plasma-derived inter-alpha-trypsin inhibitor complex, its bikunin subunit, and the second carboxyl-terminal Kunitz-type domain of bikunin were identified as genuine physiologic inhibitors with K(i) values of 64, 50, and 22 nM, respectively. Inter-alpha-trypsin inhibitor and free bikunin have the potential to neutralize extracellular granzyme K activity after T cell degranulation and may thus control unspecific damage of bystander cells at sites of inflammatory reactions.

A possible prebiotic synthesis of pantetheine, a precursor to coenzyme A.

The involvement of coenzyme A in many enzyme reactions suggests that it acted in this capacity very early in the development of life on Earth. Particularly relevant in this regard is its role in the activation of amino acids and hydroxy acids in the biosynthesis of some peptide antibiotics--a mechanism of peptide synthesis that forms the basis for the proposal that a thioester world could have preceded the RNA world. The components of coenzyme A have been shown to be probable prebiotic compounds: beta-alanine, pantoyl lactone and cysteamine and possibly adenosine. We show here that the pantetheine moiety of coenzyme A (which also occurs in a number of enzymes) can be synthesized in yields of several per cent by heating pantoyl lactone, beta-alanine and cysteamine at temperatures as low as 40 degrees C. These components are extremely soluble and so would have been preferentially concentrated in evaporating bodies of water, for example on beaches and at lagoon margins. Our results show that amide bonds can be formed at temperatures as low as 40 degrees C, and provide circumstantial support for the suggestion that pantetheine and coenzyme A were important in the earliest metabolic systems.

Fluorescent probes as tools to assess the receptor for the urokinase-type plasminogen activator on tumor cells.

Flow cytofluorometric protocols (FACScan) are described for the rapid and quantitative real-time analysis of binding of FITC-pro-u-PA to cell surface receptors (u-PAR) on living, resting, and also on PMA-stimulated human monocytic U937 cells. Binding of pro-u-PA was visualized by CLSM. This fairly new technique is superior over conventional fluorescence microscopy and is an alternative to electron microscopic approaches. Both flow cytofluorometry and confocal laser scanning microscopy allow the analysis quantitatively and with high-sensitivity binding of FITC-pro-u-PA to single suspended or adherent cells. By CLSM u-PA/u-PAR were found to be located in heterogeneously distributed discrete patches at the cell surface on U937 and not inside the cell. This is in agreement with previous studies by Hansen et al, who applied radioiodinated u-PA and electron microscopy to locate u-PAR on microvilli of fixed U937 cells. By flow cytofluorometry, it was possible to quantify the time-dependent and temperature-dependent binding of FITC-pro-u-PA to living single U937. Apparent saturation of u-PAR was achieved at 5 nM FITC-pro-u-PA for both nonstimulated and PMA-stimulated U937 cells. Half saturation of u-PAR was also determined. Nonstimulated U937 was 0.7 nM, and PMA-stimulated U937 was 1.1 nM of FITC-pro-u-PA. This increase in half-saturation concentration in PMA-stimulated cells is paralleled by a steep increase in binding sites (3.6-fold). The use of fluoresceinated reference beads is recommended to verify changes in affinity and binding sites. Using CLSM or flow cytofluorometry, it is also possible to study the structure relationship of u-PA/u-PAR in the presence of competitive binding analogues or inhibitors. Fluorescence techniques will also permit the identification of u-PAR-positive cells in blood, ascitic fluid, or biopsies obtained from cancer patients.

Synthesis of the pro-peptide of subtilisin BPN'.

Subtilisin, a bacterial serine protease, is secreted as pre-pro-subtilisin. Previously, we demonstrated that the pro-peptide moiety of intact pro-subtilisin can guide the folding of inactive protein to active enzyme both in an intramolecular (6) and intermolecular manner (18). Herein is reported the total chemical synthesis of the pro-sequence (77 amino acids) of pre-pro-subtilisin BPN' carried out by solid phase methods. The structure was confirmed by both sequencing and amino acid analysis of the fragment peptides resulting from a V-8 protease digest. Preliminary studies indicate that the synthetic pro-peptide itself can renature denatured subtilisin BPN'. This study demonstrates a novel method for examining protein folding with the aid of exogenously added synthetic peptides.

Enzymic synthesis of lignin precursors. Comparison of cinnamoyl-CoA reductase and cinnamyl alcohol:NADP+ dehydrogenase from spruce (Picea abies L.) and soybean (Glycine max L.).

Cambial sap of spruce (Picea abies) proved to be a good source for isolation of cinnamoyl-CoA reductase and cinnamyl alcohol:NADP+ dehydrogenase. Apparently homogeneous enzymes were obtained by a multistep procedure including dye-ligand chromatography and for the reductase also affinity chromatography on (coenzyme A)-agarose. An improved purification procedure for the reductase from soybean cell cultures is also reported. Molecular weights and subunit composition of reductase and dehydrogenase from spruce are very similar to those of the corresponding enzymes from soybean. Reduction of feruloyl-CoA to coniferaldehyde catalysed by the reductase is a freely reversible reaction with an equilibrium constant of 5.6 x 10(-4) M at pH 6.25. A strong dependence of the Michaelis constants on the type of buffer was found. For reductase the Km-value of feruloyl-CoA in phosphate buffer (5.2 microM) is about 14-times similar than in citrate buffer (73 microM). Pronounced differences in substrate specificities between the enzymes from spruce and soybean were found, which reflect the different lignin composition of gymnosperms and dicotyledenous angiosperms. From the kinetic constants of the enzymes it can be concluded that under physiological conditions feruloyl-CoA is the preferred substrate for the reductase from both sources whereas sinapoyl-CoA is a substrate only for the soybean reductase and sinapyldehyde a substrate only for the soybean dehydrogenase. 4-Coumaroyl-CoA is a poor substrate for the reductase from both spruce and soybean. This result is consistent with the low content of 4-coumaryl alcohol units in gymnosperm and angiosperm lignin.