Polyketides, the ubiquitous products of secondary metabolism in microorganisms, are made by a process resembling fatty acid biosynthesis that allows the suppression of reduction or dehydration reactions at specific biosynthetic steps, giving rise to a wide range of often medically useful products. The lovastatin biosynthesis cluster contains two type I polyketide synthase genes. Synthesis of the main nonaketide-derived skeleton was found to require the previously known iterative lovastatin nonaketide synthase (LNKS), plus at least one additional protein (LovC) that interacts with LNKS and is necessary for the correct processing of the growing polyketide chain and production of dihydromonacolin L. The noniterative lovastatin diketide synthase (LDKS) enzyme specifies formation of 2-methylbutyrate and interacts closely with an additional transesterase (LovD) responsible for assembling lovastatin from this polyketide and monacolin J.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite2. Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines2, including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17–IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI–IκBζ regulatory axis could be an important new strategy for the treatment of IL-17–IκBζ-mediated autoimmune diseases.

The presence of variable static hemin orientational disorder about the ␣-␥-meso axis in the substrate complexes of mammalian heme oxygenase, together with the incomplete averaging of a second, dynamic disorder, for each hemin orientation, has led to NMR spectra with severe spectral overlap and loss of key two-dimensional correlations that seriously interfere with structural characterization in solution. We demonstrate that the symmetric substrate, 2,4-dimethyldeuterohemin, yields a single solution species for which the dynamic disorder is sufficiently rapid to allow effective and informative Mammalian heme oxygenase (HO)1 is a ϳ300-residue, membrane-bound, non-heme enzyme that, using heme as cofactor and substrate, catalyzes the regiospecific conversion of heme to ␣-biliverdin, iron, and CO (1). The physiological roles of HO are heme catabolism (HO-1) (2-4) and the generation of CO as a putative neural messenger (HO-2) (5, 6). Detailed mechanistic (7-13) and spectroscopic (13-17) studies of the fully active recombinant, soluble 265-residue portion of HO-1 have shown that, in contrast to heme peroxidase and cytochrome P450, HO does not act through a ferryl intermediate. Recent crystal structures (18,19) of the substrate-bound, water-ligated complexes of a more truncated 233-residue human HO, hHO (20), and the complete rat HO (18), rHO, have revealed a largely helical enzyme that confirms the binding of heme by His-25 and locates a highly bent distal helix that is sufficiently close to the heme to sterically block all but the ␣-meso position (see Fig.

The fungal metabolite lovastatin (1) 1 and its derivatives are cholesterol-lowering drugs that act as potent inhibitors of (3S)hydroxy-3-methylglutaryl-coenzyme A reductase. 2 Although 1 and compactin 3 have attracted attention from synthetic chemists, 4 these drugs and some analogues (e.g., simvastatin, pravastatin) which are used in humans are manufactured by fermentation, either directly or with subsequent chemical or microbial modification. Studies on the biosynthesis of 1 in Aspergillus terreus indicate that it is formed by a polyketide pathway. [5][6][7] Of special interest is the proposal of an enzyme-catalyzed Diels-Alder cyclization of the intermediate hexaketide triene to generate the decalin system (Figure 1). 7,8 This idea is supported by the formation of dihydromonacolin L (2) 9 by a heterologous host, A. nidulans containing the loVB and loVC genes from A. terreus. 10,11 Corresponding heterologous expression of the loVB protein (lovastatin nonaketide synthase, LNKS) without loVC leads to truncated pyrones 3 and 4, formed due to failure of enoyl reduction at the tetraketide stage. 10 There are proposals that enzyme-catalyzed Diels Alder reactions may occur during biosynthesis of many secondary metabolites, 12 but the demonstrated ability of pure biological macromolecules to promote this process has been limited to catalytic antibodies generated from synthetic haptens 13 and to synthetic RNA fragments that bind metals. 14 There is also a report of a crude cellfree preparation from the fungus Alternaria solani that oxidizes an achiral allylic alcohol, prosolanopyrone II, to a conjugated triene aldehyde, thereby triggering intramolecular Diels-Alder cyclization to an optically active product, solanopyrone A. 15 We now report that purified LNKS catalyzes intramolecular Diels-Alder endo closure of a substrate analogue, (E,E,E)-(R)-6-methyldodecatri-2,8,10-enoic acid N-acetylcysteamine (NAC) thioester (5), to a bicyclic system with the same ring stereochemistry as 2, which is different from that obtained in nonenzymatic cyclization.

Upon activation, macrophages undergo extensive metabolic rewiring 1 , 2 . Production of itaconate through the inducible enzyme IRG1 is a key hallmark of this process 3 . Itaconate inhibits succinate dehydrogenase (SDH) 4 , 5 , has electrophilic properties 6 , and is associated with a change in cytokine production 4 . Here, we compare the metabolic, electrophilic, and immunologic profiles of macrophages treated with unmodified itaconate and a panel of commonly used itaconate derivatives to examine its role. Using wild type and Irg1 −/− macrophages, we show that neither dimethyl itaconate (DI), 4-octyl itaconate (4OI), nor 4-monoethyl itaconate (4EI) are converted into intracellular itaconate, while exogenous itaconic acid readily enters macrophages. We find that only DI and 4OI induce a strong electrophilic stress response, in contrast to itaconate and 4EI. This correlates with their immunosuppressive phenotype: DI and 4OI inhibit IκBζ and pro-IL-1β induction, as well as IL-6, IL-10, and IFN-β secretion in an Nrf2-independent manner. In contrast, itaconate treatment only suppressed IL-1β secretion but not pro-IL-1β levels, and, surprisingly, strongly enhanced LPS-induced IFN-β secretion. Consistently, Irg1 −/− macrophages produced lower levels of interferon and reduced transcriptional activation of this pathway. Our work establishes itaconate as an immunoregulatory, rather than strictly immunosuppressive metabolite, and highlights the importance of using unmodified itaconate in future studies.

Norcarane (1) and spiro[2.5]octane (2) yield different product distributions depending on whether they are oxidized via concerted, radical, or cationic mechanisms. For this reason, these two probes were used to investigate the mechanisms of hydrocarbon hydroxylation by two mammalian and two bacterial cytochrome P450 enzymes. Products indicative of a radical intermediate with a lifetime ranging from 16 to 52 ps were detected during the oxidation of norcarane by P450(cam) (CYP101), P450(BM3) (CYP102), CYP2B1, and CYP2E1. Trace amounts of the cation rearrangement product were observed with norcarane for all but CYP2E1, while no cation or radical rearrangement products were observed for spiro[2.5]octane. The results for the oxidation of norcarane with a radical rearrangement rate of 2 x 10(8) s(-1) are consistent with the involvement of a two-state radical rebound mechanism, while for the slower (5 x 10(7) s(-1)) spiro[2,5]oct-4-yl radical rearrangement products were beyond detection. Taken together with earlier data for the hydroxylation of bicyclo[2.1.0]pentane, which also suggested a 50 ps radical lifetime, these three structurally similar and functionally simple substrates show a consistent pattern of rearrangement that supports a radical rebound mechanism for this set of cytochrome P450 enzymes.

To examine the roles of the proximal thiolate iron ligand, the C357H mutant of P450(cam) (CYP101) was characterized by resonance Raman, UV, circular dichroism, and activity measurements. The C357H mutant must be reconstituted with hemin for activity to be observed. The reconstituted enzyme is a mixture of high and low spin species. Low temperature (10 degrees C), low enzyme concentration (1 microM), high camphor concentration (1 mM), and 5--50 mM buffer concentrations increase the high to low spin ratio, but under no conditions examined was the protein more than 60% high spin. The C357H mutant has a poorer K(m) for camphor (23 vs 2 microM) and a poorer K(d) for putidaredoxin (50 vs 20 microM) than wild-type P450(cam). The mutant also exhibits a greatly decreased camphor oxidation rate, elevated uncoupling rate, and much greater peroxidase activity. Electron transfer from putidaredoxin to the mutant is much slower than to the wild-type even though redox potential measurements show that the electron transfer remains thermodynamically favored. These experiments confirm that the thiolate ligand facilitates the O--O bond cleavage by P450 enzymes and also demonstrate that this ligand satisfies important roles in protein folding, substrate binding, and electron transfer.

Mechanochemistry enables enzymatic cleavage of cellulose into glucose without bulk solvents, acids, other aggressive reagents, or substrate pre-treatment. This clean mechanoenzymatic process (coined RAging) is also directly applicable to biomass, avoids many limitations associated with the use of cellulases, and produces glucose concentrations greater than three times that obtained by conventional methods.