13 C{2 H} rotational echo double resonance NMR has been used to provide the first evidence for the formation of quinone-derived cross-links in mussel byssal plaques. Labeling of byssus was achieved by allowing mussels to filter feed from seawater containing L-[phenol-4-13 C]tyrosine and L-[ring-d 4 ]tyrosine for 2 days. Plaques and threads were harvested from two groups of mussels over a period of 28 days. One group was maintained in stationary water while the other was exposed to turbulent flow at 20 cm/s. The flow-stressed byssal plaques exhibited significantly enhanced levels of 5, 5-di-dihydroxyphenylalanine cross-links. The average concentration of di-dihydroxyphenylalanine cross-links in byssal plaques is 1 per 1800 total protein amino acid residues.The attachment strategies of marine organisms that rely on DOPA-containing 1 adhesive proteins have recently come under much scrutiny. A number of these organisms, including tubebuilding polychaetes, black corals, ascidians, and mussels, cure their proteinaceous adhesives by a process called "quinone tanning" (1). Despite frequent references to in vitro studies postulating that quinone tanning involves nucleophilic addition of amine groups to quinones (2), nothing of substance is known about the actual cross-linking chemistry in these organisms. Indeed, previous efforts from our own laboratories seemed to rule out the existence of lysine-aromatic or aromaticaromatic coupling products in the mussel byssus (3, 4). In 1994, Dolmer and Svane (5) reported that individual threads in a mussel byssus behaved as "smart materials" in flow: by doubling the flow of seawater, the tensile strength of the threads could be doubled. This suggested that there were different degrees of quinone-tanning in a given material that relied in some way on maturation by imposed stresses. This paper reports on the continuation of work on the biosynthetic labeling of byssus with 13 C-and 2 H-containing analogs of tyrosine and the in situ analysis of this label-rich material by rotational echo double resonance (REDOR) NMR (6). We have repeated the byssus labeling under high flow conditions and compared directly REDOR spectra of byssal plaques collected under stressed (flow) and unstressed (stationary or minimal flow) conditions. The difference between these REDOR spectra reveal the preferential routing of tyrosine labels to diphenolics. The 13 C{ 2 H} REDOR dephasing is only consistent with the formation of 5, 5Ј-di-DOPA covalent crosslinks stabilizing the byssal plaques formed under stress. EXPERIMENTAL PROCEDURESMussel Labeling-170 Adult mussels (Mytilus edulis) were collected in the vicinity of Roosevelt Inlet at Lewes, Delaware. The mussels ranged in size from 3 to 10 cm. They were tethered to acrylic plates as described previously (4). The plates were put into two 140-liter marine aquarium tanks with salt water (no flow) at 14°C. The tanks were aerated. Labels H 4 ]tyrosine. The mussels were incubated with the labels for 48 h, after which control samples of plaques and threads were harv...

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3-phosphoadenosine 5-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates. Heparan sulfate (HS)3 is a ubiquitous component of the cell surface and extracellular matrix. It regulates a wide range of physiologic and pathophysiologic functions, including embryonic development and blood coagulation and can facilitate viral infection (1, 2). HS exerts its biological effects by interacting with the specific proteins involved in a given process (3). HS is a highly charged polysaccharide consisting of 1 3 4-linked glucosamine and glucuronic/iduronic acid units that contain both N-and O-sulfo groups. Unique saccharide sequences within HS determine the specificity of the binding of HS to its target proteins (4). Heparin, a specialized form of HS, is a commonly used anticoagulant drug. Thus, new methods for the synthesis of heparin and HS attract considerable interest for those developing anticoagulant drugs having improved pharmacological effects.Chemical synthesis has been the major route to obtain structurally defined heparin and HS oligosaccharides (5). The most important example involves the structure antithrombin-binding pentasaccharide. A synthetic pentasaccharide, based on this structure, has been marketed in the United States under the trade name Arixtra. Arixtra is a specific factor Xa inhibitor that is used clinically to prevent venous thromboembolic incidents during surgery. Unfortunately, the total synthesis of heparin and HS oligosaccharides, larger than pentasaccharides, is extremely difficult. HS analogues with 14 saccharide units inhibit the activity of thrombin, but these synthetic analogues are simplified hybrid molecules of HS oligosaccharides and highly sulfated glucose units (6, 7) and are not the naturally occurring structures. Although our laboratory and others continue to pursue the synthesis of heparin and HS oligosaccharides (8), it has become clear that chemical synthesis alone is currently incapable of generating most larger oligosaccharide structures. The application of HS biosynthetic enzymes for generating large heparin and HS oligosaccharides with desired biological activities o...

No abstract

No abstract

The 46-kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP. The reaction is inhibited by N-(phosphonomethyl)glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, we have used dipolar recovery at the magic angle (DRAMA) and rotational-echo double resonance (REDOR) to determine intra- and interligand internuclear distances. DRAMA was used to determine the single 31P-31P distance, while REDOR was used to determine one 31P-15N distance and five 31P-13C distances. These experimental distances were used as restraints in molecular dynamics simulations of an S3P-Glp complex to examine the geometry of the two ligands relative to one another in the ternary complex. The simulations were compared to unrestrained simulations of the EPSP synthase tetrahedral intermediate and its phosphonate analog. The results suggest that Glp is unlikely to bind in the same fashion as PEP, a conclusion that is consistent with recent studies that have questioned the role of Glp as a transition-state or intermediate analog.