Abstract
The zinc insulin hexamer undergoes allosteric reorganization among three conformational states, designated T(6), T(3)R(3)(f), and R(6). Although the free monomer in solution (the active species) resembles the classical T-state, an R-like conformational change is proposed to occur upon receptor binding. Here, we distinguish between the conformational requirements of receptor binding and the crystallographic TR transition by design of an active variant refractory to such reorganization. Our strategy exploits the contrasting environments of His(B5) in wild-type structures: on the T(6) surface but within an intersubunit crevice in R-containing hexamers. The TR transition is associated with a marked reduction in His(B5) pK(a), in turn predicting that a positive charge at this site would destabilize the R-specific crevice. Remarkably, substitution of His(B5) (conserved among eutherian mammals) by Arg (occasionally observed among other vertebrates) blocks the TR transition, as probed in solution by optical spectroscopy. Similarly, crystallization of Arg(B5)-insulin in the presence of phenol (ordinarily a potent inducer of the TR transition) yields T(6) hexamers rather than R(6) as obtained in control studies of wild-type insulin. The variant structure, determined at a resolution of 1.3A, closely resembles the wild-type T(6) hexamer. Whereas Arg(B5) is exposed on the protein surface, its side chain participates in a solvent-stabilized network of contacts similar to those involving His(B5) in wild-type T-states. The substantial receptor-binding activity of Arg(B5)-insulin (40% relative to wild type) demonstrates that the function of an insulin monomer can be uncoupled from its allosteric reorganization within zinc-stabilized hexamers.
Highlights
Polyhydroxyalkanoate (PHA) biopolymers are widespread among microbes and are used for storage of carbon and reducing equivalents in intracellular granules [45]
We describe reconstitution of the novel propionyl-CoA biosynthesis pathway (Sbm and YgfG activities) in a recombinant strain of S. enterica that has a mutation in prpC and harbors the Acinetobacter PHA synthesis operon
S. enterica JE4199 harboring pISA1 clearly showed enhanced HV incorporation in the copolymer compared to the incorporation in the parent when propionate was added to the medium, indicating that when the 2-methylcitric acid cycle was blocked, more propionyl-CoA was shunted to HV biosynthesis (Table 2)
Summary
Polyhydroxyalkanoate (PHA) biopolymers are widespread among microbes and are used for storage of carbon and reducing equivalents in intracellular granules [45] These natural polyesters have attracted considerable attention because they have properties similar to those of common thermoplastics or elastomers, depending on the monomeric composition. Biopol [poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)], a copolymer of monomers of 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV), is a biodegradable PHA thermoplastic that was produced by Imperial Chemical Industries, Zeneca Bio Products, and Monsanto [3, 7, 33]. The latter company terminated production at the end of 1998 because of high production costs. A more economical alternative is to produce propionyl-CoA from an inexpensive, unrelated carbon source
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