Novel sol-gel synthetic techniques were used to immobilize copper-zinc superoxide dismutase (CuZnSOD), cytochrome c, and myoglobin (Mb) by encapsulation in stable, optically transparent, porous silica glass matrices under mild conditions such that the biomolecules retained their characteristic reactivities and spectroscopic properties. The resulting glasses allowed transport of small molecules into and out of the glasses at reasonable rates but nevertheless retained the protein molecules within their pores. Chemical reactions of the immobilized proteins could be monitored by means of changes in their visible absorption spectra. Silica glasses containing the immobilized proteins were observed to have similar reactivities and spectroscopic properties to those found for the proteins in solution. For example, encapsulated CuZnSOD was demetallated and remetallated, encapsulated ferricytochrome c was reduced and then reoxidized, and encapsulated met Mb was reduced to deoxy Mb and then reacted either with dioxygen to make oxy Mb or with carbon monoxide to make carbonyl Mb.

AQ4N [1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione], a prodrug with two dimethylamino N-oxide groups, is converted to the topoisomerase II inhibitor AQ4 [1,4-bis{[2-(dimethylamino)ethyl]amino}-5,8-dihydroxy-anthracene-9,10-dione] by reduction of the N-oxides to dimethylamino substituents. Earlier studies showed that several drug-metabolizing cytochrome P450 (P450) enzymes can catalyze this reductive reaction under hypoxic conditions comparable with those in solid tumors. CYP2S1 and CYP2W1, two extrahepatic P450 enzymes identified from the human genome whose functions are unknown, are expressed in hypoxic tumor cells at much higher levels than in normal tissue. Here, we demonstrate that CYP2S1, contrary to a published report (Mol Pharmacol 76:1031-1043, 2009), is efficiently reduced by NADPH-P450 reductase. Most importantly, both CYP2S1 and CYP2W1 are better catalysts for the reductive activation of AQ4N to AQ4 than all previously examined P450 enzymes. The overexpression of CYP2S1 and CYP2W1 in tumor tissues, together with their high catalytic activities for AQ4N activation, suggests that they may be exploited for the localized activation of anticancer prodrugs.

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The primary sequences of the three mammalian nitric-oxide synthase (NOS) isoforms differ by the insertion of a 52-55-amino acid loop into the reductase domains of the endothelial (eNOS) and neuronal (nNOS), but not inducible (iNOS). On the basis of studies of peptide derivatives as inhibitors of ⅐ NO formation and calmodulin (CaM) binding (Salerno, J. C., Harris, D. E., Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Martasek, P., Roman, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q., and Gross, S. S. (1997) J. Biol. Chem. 272, 29769 -29777), the insert has been proposed to be an autoinhibitory element. We have examined the role of the insert in its native protein context by deleting the insert from both wild-type eNOS and from chimeras obtained by swapping the reductase domains of the three NOS isoforms. The Ca 2؉ concentrations required to activate the enzymes decrease significantly when the insert is deleted, consistent with suppression of autoinhibition. Furthermore, removal of the insert greatly enhances the maximal activity of wild-type eNOS, the least active of the three isoforms. Despite the correlation between reductase and overall enzymatic activity for the wild-type and chimeric NOS proteins, the loop-free eNOS still requires CaM to synthesize ⅐ NO. However, the reductive activity of the CaM-free, loop-deleted eNOS is enhanced significantly over that of CaM-free wild-type eNOS and approaches the same level as that of CaMbound wild-type eNOS. Thus, the inhibitory effect of the loop on both the eNOS reductase and ⅐ NO-synthesizing activities may have an origin distinct from the loop's inhibitory effects on the binding of CaM and the concomitant activation of the reductase and ⅐ NO-synthesizing activities. The eNOS insert not only inhibits activation of the enzyme by CaM but also contributes to the relatively low overall activity of this NOS isoform.The enzymatic activities of the three NOS 1 isoforms (1-7) differ in their Ca 2ϩ -dependence: nNOS (NOS-I) and eNOS (NOS-III) are Ca 2ϩ -dependent constitutive isoforms, whereas iNOS (NOS-II), as typified by the inducible macrophage and hepatocyte form, is essentially Ca 2ϩ -independent. This difference in the Ca 2ϩ dependence of the NOS isoforms is the result of a Ca 2ϩ requirement for the reversible binding of CaM to the constitutive isoforms (8, 9), in contrast to the almost Ca 2ϩ -independent, high affinity binding of CaM to the inducible isoform (10).The NOS isoforms are also differentiated by their maximum enzymatic activity, nNOS (8,(11)(12)(13)(14) and iNOS (15-18) exhibiting much higher overall activities than eNOS (19 -24). We have shown that the lower activity of eNOS is caused by a lower ability of its flavoprotein reductase domain to transfer electrons to the catalytic heme domain (25). However, the structural features that impair the reductase activity in eNOS, and hence lower the overall catalytic activity, remain unknown.Recent evidence (26) suggests that the Ca 2ϩ dependence of nNOS and eNOS is caused by the pr...

Epothilones are potential anticancer drugs that stabilize microtubules by binding to tubulin in a manner similar to paclitaxel. Cytochrome P450epoK (P450epoK), a heme containing monooxygenase involved in epothilone biosynthesis in the myxobacterium Sorangium cellulosum, catalyzes the epoxidation of epothilones C and D into epothilones A and B, respectively. The 2.10-, 1.93-, and 2.65-Å crystal structures reported here for the epothilone D-bound, epothilone B-bound, and substratefree forms, respectively, are the first crystal structures of an epothilone-binding protein. Although the substrate for P450epoK is the largest of a P450 whose x-ray structure is known, the structural changes along with substrate binding or product release are very minor and the overall fold is similar to other P450s. The epothilones are positioned with the macrolide ring roughly perpendicular to the heme plane and I helix, and the thiazole moiety provides key interactions that very likely are critical in determining substrate specificity. Interestingly, there are strong parallels between the epothilone/P450epoK and paclitaxel/tubulin interactions. Based on structural similarities, a plausible epothilone tubulin-binding mode is proposed.Since the discovery of paclitaxel (an active ingredient of Taxol®) from Taxus brevifolia (1) and its clinical success as an anti-cancer drug, there have been extensive efforts to find compounds with similar action. Those efforts resulted in the identification of three other classes of compounds from natural sources: epothilones, produced by the cellulose-degrading myxobacterium Sorangium cellulosum (2), the marine spongederived discodermolide (3, 4), and the coral-derived eleutherobins (5)/sarcodictyins (6) (Fig. 1). All three classes, like paclitaxel, bind to and stabilize microtubules leading to mitotic arrest of the cycle at G 2 -M phase and subsequently induction of cell death in several cell lines (7,8). Epothilones, however, offer some advantages, because they are effective against Pglycoprotein-expressing multidrug-resistant cell lines, are active in a cell line with paclitaxel resistance (7), and the water solubility of epothilones is significantly greater than that of paclitaxel. Another advantage is that epothilones can be produced in large quantities using a heterologous expression system (9).One step in the biosynthesis of epothilones involves a C12-C13 epoxidation (Fig. 1a) by a cytochrome P450, P450epoK (9). Cytochrome P450s (P450s) 1 are heme-containing monooxygenases best known for their role in drug detoxification (10). However, P450s also are involved in steroid hormone biosynthesis (11) as well as the biosynthesis of important macrolide antibiotics like erythromycin (12) and rapamycin (13). To date, there is no known protein structure complexed with an epothilone. In addition, epothilone represents the largest substrate of a P450 where the crystal structure of the enzyme-substrate complex is known and thus provides important insights in understanding how P450 architecture adapts to the r...

We have investigated factors that influence the properties of the zinc binding site in yeast copper-zinc superoxide dismutase (CuZnSOD). The properties of yeast CuZnSOD are essentially invariant from pH 5 to pH 9. However, below this pH range there is a change in the nature of the zinc binding site which can be interpreted as either (1) a change in metal binding affinity from strong to weak, (2) the expulsion of the metal bound at this site, or (3) a transition from a normal distorted tetrahedral ligand orientation to a more symmetric arrangement of ligands. This change is strongly reminiscent of a similar pH-induced transition seen for the bovine protein and, based on the data presented herein, is proposed to be a property that is conserved among CuZnSODs. The transition demonstrated for the yeast protein is not only sensitive to the pH of the buffering solution but also to the occupancy and redox status of the adjacent copper binding site. Furthermore, we have investigated the effect of single site mutations on the pH- and redox-sensitivity of Co2+ binding at the zinc site. Each of the mutants H46R, H48Q, H63A, H63E, H80C, G85R, and D83H is capable of binding Co2+ to a zinc site with a distorted tetrahedral geometry similar to that of wild-type. However, they do so only if Cu+ is bound at the copper site or if the pH in raised to near physiological levels, indicating that the change at the zinc binding site seen in the wild-type is conserved in the mutants, albeit with an altered pKa. The mutants H71C and D83A did not bind Co2+ in a wild-type-like fashion under any of the conditions tested. This study reveals that the zinc binding site is exquisitely sensitive to changes in the protein environment. Since three of the mutant yeast proteins investigated here contain mutations analogous to those that cause ALS (amyotrophic lateral sclerosis) in humans, this finding implicates improper metal binding as a mechanism by which CuZnSOD mutants exert their toxic gain of function.

The nitric oxide synthases (NOS) are single polypeptides that encode a heme domain, a calmodulin binding motif, and a flavoprotein domain with sequence similarity to P450 reductase. Despite this basic structural similarity, the three major NOS isoforms differ significantly in their rates of ⅐NO synthesis, cytochrome c reduction, and NADPH utilization and in the Ca 2؉ dependence of these rates. To assign the origin of these differences to specific protein domains, we constructed chimeras in which the reductase domains of endothelial and inducible NOS, respectively, were replaced by the reductase domain of neuronal NOS. The results with the chimeric proteins confirm the modular organization of the NOS polypeptide chain and demonstrate that (a) similar residues establish the necessary contacts between the reductase and heme domains in the three NOS isoforms, (b) the maximal rate of ⅐NO synthesis is determined by the maximum intrinsic ability of the reductase domain to deliver electrons to the heme domain, (c) the Ca The flavin domain uncouples the electrons provided by NADPH and delivers them, one at a time, to the prosthetic heme iron atom. H 4 B is required for ⅐NO synthesis by all three proteins, but its role in the catalytic process remains unclear (9).Clear differences exist in the Ca 2ϩ and CaM dependence of the three NOS isoforms. The binding of CaM to nNOS and eNOS, the two constitutive isoforms, is Ca 2ϩ -dependent and reversible (10, 11), whereas the binding of CaM to iNOS is essentially a Ca 2ϩ -independent, irreversible process (12). Thus, the catalytic activities of nNOS and eNOS are regulated by cellular Ca 2ϩ -levels, whereas the activity of iNOS is insensitive to the Ca 2ϩ concentration and is primarily regulated by the rate at which the protein is synthesized (12). In addition to the heme, CaM, and reductase domains common to all the isoforms, nNOS has an additional N-terminal domain thought to be involved in subcellular targeting (13,14), and eNOS is unique in that it has myristoylation and palmitoylation sites that target it to the membrane (15, 16).The catalytic activities of the NOS isoforms differ considerably, regardless of whether activity is measured as ⅐NO formation or cytochrome c reduction. Regardless of which of these two parameters is measured, the activities of iNOS and nNOS are considerably higher than that of eNOS. Thus, in our hands, the V max values for ⅐NO formation by recombinant iNOS (17), nNOS (18), and eNOS (19) were found to be ϳ800, ϳ400, and ϳ130 nmol min Ϫ1 mg Ϫ1 , respectively, and the rates of electron transfer to cytochrome c in the presence of CaM and Ca 2ϩ were ϳ45,000, ϳ44,000, and ϳ1,800 nmol min Ϫ1 mg Ϫ1 , respectively. In each case, the reductase domain was able to transfer electrons to cytochrome c at a rate much greater than the maximum rate of ⅐NO production, although the nature of the ratelimiting step(s) in ⅐NO formation and the reason(s) for the differences in the catalytic activities of the isoforms are unclear. The reduction of cytochrome c by nNOS and eN...

The epothilones are a new class of highly promising anticancer agents with a mode of action akin to that of paclitaxel but with distinct advantages over that drug. The principal natural compounds are epothilones A and B, which have an epoxide in the macrocyclic lactone ring, and C and D, which have a double bond instead of the epoxide group. The epoxidation of epothilones C and D to A and B, respectively, is mediated by EpoK, a cytochrome P450 enzyme encoded in the epothilone gene cluster. Here we report high-yield expression of EpoK, characterization of the protein, demonstration that the natural substrate can prevent-and even reverse-denaturation of the protein, identification of ligands and surrogate substrates, development of a high-throughput fluorescence activity assay based on the H(2)O(2)-dependent oxidation of 7-ethoxy-4-trifluoromethylcoumarin, and identification of effective inhibitors of the enzyme. These results will facilitate improvements in the yields of epothilones C and D and the engineering of EpoK to prepare novel epothilone analogues. Furthermore, the finding that the denatured enzyme is rescued by the substrate offers a potential paradigm for control of the P450 catalytic function.