A low pKa cysteine at the active site of mouse methionine sulfoxide reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. Recently it has also been shown to catalyze the reverse reaction, oxidizing methionine residues to methionine sulfoxide. A cysteine at the active site of the enzyme is essential for both reductase and oxidase activities. This cysteine has been reported to have a pK(a) of 9.5 in the absence of substrate, decreasing to 5.7 upon binding of substrate. Using three independent methods, we show that the pK(a) of the active site cysteine of mouse methionine sulfoxide reductase is 7.2 even in the absence of substrate. The primary mechanism by which the pK(a) is lowered is hydrogen bonding of the active site Cys-72 to protonated Glu-115. The low pK(a) renders the active site cysteine susceptible to oxidation to sulfenic acid by micromolar concentrations of hydrogen peroxide. This characteristic supports a role for methionine sulfoxide reductase in redox signaling.     

Identification of active site residues of Escherichia coli fumarate reductase by site-directed mutagenesis.

Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor. The enzyme is structurally and catalytically similar to succinate dehydrogenase (succinate-ubiquinone oxidoreductase) from both procaryotes and eucaryotes. Both enzymes have been proposed to contain an essential cysteine residue at the active site based on studies with thiol-specific reagents. Chemical modification studies have also suggested roles for essential histidine and arginine residues in catalysis by succinate dehydrogenase. In the present study, a combination of site-directed mutagenesis and chemical modification techniques have been used to investigate the role(s) of the conserved histidine 232, cysteine 247, and arginine 248 residues of the flavorprotein subunit (FrdA) in active site function. A role for His-232 and Arg-248 of FrdA is shown by loss of both fumarate reductase and succino-oxidase activities following site-directed substitution of these particular amino acids. Evidence is also presented that suggests a second arginine residue may form part of the active site. Potential catalytic and substrate-binding roles for arginine are discussed. The effects of removing histidine-232 of FrdA are consistent with its proposed role as a general acid-base catalyst. The fact that succinate oxidation but not fumarate reduction was completely lost, however, might suggest that alternate proton donors substitute for His-232. The data confirm that cysteine 247 of FrdA is responsible for the N-ethylmaleimide sensitivity shown by fumarate reductase but is not required for catalytic activity or the tight-binding of oxalacetate, as previously thought.     

Inactivation of rat testicular NADPH-cytochrome P-450 reductase by 2,4,6-trinitrobenzenesulfonate

Rat testicular NADPH-cytochrome P-450 reductase was inactivated by treatment with 2,4,6-trinitrobenzene sulfonate (TNBS) or with 2',3'-dialdehyde derivatives of 5'-ATP and NADP+. The inactivation rates were dependent on reaction time and followed pseudo-first order kinetics. The rate of inactivation of cytochrome c reducing activity by TNBS was faster than that of reducing activities for K3Fe(CN)6 and for dichlorophenol indophenol (DCPIP). Cytochrome c and DCPIP prevented NADPH-cytochrome P-450 reductase from inactivation by TNBS, but NADP(H) protected to a lesser extent. Stoichiometry indicated that two residues of amino acid modified with TNBS were essential for the enzyme activity. The 2',3'-dialdehyde derivatives of 5'-ATP and NADP+ were specific ligands for the modification of lysine residues, whereas TNBS would possibly modify residues of lysine and/or cysteine. By differential and sequential modification by 5,5'-dithio-bis(2-nitrobenzoic acid), TNBS and dithiothreitol, the residues of lysine and cysteine were identified in the active site of NADPH-cytochrome P-450 reductase. These results suggest that lysyl and cysteinyl residues are located at or near the active region of NADPH-cytochrome P-450 reductase from the rat testicular microsomal fraction.     

Active site of mercuric reductase resides at the subunit interface and requires Cys135 and Cys140 from one subunit and Cys558 and Cys559 from the adjacent subunit: evidence from in vivo and in vitro heterodimer formation

Mercuric reductase catalyzes the two-electron reduction of Hg(II) to Hg(0) using NADPH as the reductant; this reaction constitutes the molecular basis for detoxification of Hg(II) by bacteria. The enzyme is an alpha 2 homodimer and possesses two pairs of cysteine residues, Cys135 and Cys140 (redox-active pair) and Cys558 and Cys559 (C-terminal pair), which are known to be essential for catalysis. In the present study, we have obtained evidence for an intersubunit active site, consisting of a redox-active cysteine pair from one subunit and a C-terminal pair from the adjacent subunit, by reconstituting catalytic activity both in vivo and in vitro starting with two inactive, mutant enzymes, Ala135Ala140Cys558Cys559 (AACC) and Cys135Cys140Ala558Ala559 (CCAA). Genetic complementation studies were used to show that coexpression of AACC and CCAA in the same cell yielded an HgR phenotype, some 10(4)-fold more resistant than cells expressing only one mutant. Purification and catalytic characterization of a similarly coexpressed protein mixture showed the mixture to have activity levels ca. 25% those of wild type; this is the same as that statistically anticipated for a CCAA-AACC heterodimeric/homodimeric mixture with only one functional active site per heterodimer. Actual physical evidence for the formation of active mutant heterodimers was obtained by chaotrope-induced subunit interchange of inactive pure CCAA and AACC homodimers in vitro followed by electrophoretic separation of heterodimers from homodimers. Taken together, these data provide compelling evidence that the active site in mercuric reductase resides at the subunit interface and contains cysteine residues originating from separate polypeptide chains.     

Anaerobic sulfatase-maturating enzymes, first dual substrate radical S-adenosylmethionine enzymes

Sulfatases are a major group of enzymes involved in many critical physiological processes as reflected by their broad distribution in all three domains of life. This class of hydrolases is unique in requiring an essential post-translational modification of a critical active-site cysteine or serine residue to C(alpha)-formylglycine. This modification is catalyzed by at least three nonhomologous enzymatic systems in bacteria. Each enzymatic system is currently considered to be dedicated to the modification of either cysteine or serine residues encoded in the sulfatase-active site and has been accordingly categorized as Cys-type and Ser-type sulfatase-maturating enzymes. We report here the first detailed characterization of two bacterial anaerobic sulfatase-maturating enzymes (anSMEs) that are physiologically responsible for either Cys-type or Ser-type sulfatase maturation. The activity of both enzymes was investigated in vivo and in vitro using synthetic substrates and the successful purification of both enzymes facilitated the first biochemical and spectroscopic characterization of this class of enzyme. We demonstrate that reconstituted anSMEs are radical S-adenosyl-l-methionine enzymes containing a redox active [4Fe-4S](2+,+) cluster that initiates the radical reaction by binding and reductively cleaving S-adenosyl-l-methionine to yield 5 '-deoxyadenosine and methionine. Surprisingly, our results show that anSMEs are dual substrate enzymes able to oxidize both cysteine and serine residues to C(alpha)-formylglycine. Taken together, the results support a radical modification mechanism that is initiated by hydrogen abstraction from a serine or cysteine residue located in an appropriate target sequence.     

o-Phthalaldehyde as a probe in the active site of phosphoenolpyruvate carboxylase

Modification of phosphoenolpyruvate carboxylase with o-phthalaldehyde (OPA) resulted in rapid and irreversible inactivation exhibiting biphasic reaction kinetics. The kinetic analysis and correlation of spectral changes with activity indicated that inactivation by OPA results from the modification of two lysine and two cysteine residues per subunit of the enzyme. PEP plus Mg2+ offered substantial protection against modification. Some of the effectors also gave appreciable protection against modification indicating that the residues may be located at or close to the active site. Thus, the results indicate formation of two isoindoles showing the proximity of the essential lysine and cysteine residues at the active site.     

Glutathionylation of trypanosomal thiol redox proteins

Trypanosomatids, the causative agents of several tropical diseases, lack glutathione reductase and thioredoxin reductase but have a trypanothione reductase instead. The main low molecular weight thiols are trypanothione (N(1),N(8)-bis-(glutathionyl)spermidine) and glutathionyl-spermidine, but the parasites also contain free glutathione. To elucidate whether trypanosomes employ S-thiolation for regulatory or protection purposes, six recombinant parasite thiol redox proteins were studied by ESI-MS and MALDI-TOF-MS for their ability to form mixed disulfides with glutathione or glutathionylspermidine. Trypanosoma brucei mono-Cys-glutaredoxin 1 is specifically thiolated at Cys(181). Thiolation of this residue induced formation of an intramolecular disulfide bridge with the putative active site Cys(104). This contrasts with mono-Cys-glutaredoxins from other sources that have been reported to be glutathionylated at the active site cysteine. Both disulfide forms of the T. brucei protein were reduced by tryparedoxin and trypanothione, whereas glutathione cleaved only the protein disulfide. In the glutathione peroxidase-type tryparedoxin peroxidase III of T. brucei, either Cys(47) or Cys(95) became glutathionylated but not both residues in the same protein molecule. T. brucei thioredoxin contains a third cysteine (Cys(68)) in addition to the redox active dithiol/disulfide. Treatment of the reduced protein with GSSG caused glutathionylation of Cys(68), which did not affect its capacity to catalyze reduction of insulin disulfide. Reduced T. brucei tryparedoxin possesses only the redox active Cys(32)-Cys(35) couple, which upon reaction with GSSG formed a disulfide. Also glyoxalase II and Trypanosoma cruzi trypanothione reductase were not sensitive to thiolation at physiological GSSG concentrations.     

Cysteine residues at the active site of glutamine synthetase from spinach leaves

Titration of cysteine residues of spinach glutamine synthetase with 5-5' dithiobis (2-nitrobenzoic acid) indicates that there are five such residues per monomer of enzyme and that two of these five are on the surface of the molecule. The presence of substrates, or either of the competitive inhibitors methionine sulfoximine or phosphinothricin, completely protects both of the surface sulfhydryls from titration. This suggests that both are located at the active site. In the absence of Mg2+ and ATP, both surface sulfhydryls must be modified before loss of activity. We conclude that while both of the cysteine residues are located at the active site, only one of them may be involved in catalysis. Because the cysteine residue which is implicated in catalysis can be protected by Mg2+ and ATP, we believe that it may be located at or near the binding site of these ligands.     

Diverse Functionalization of Aurora-A Kinase at Specified Surface and Buried Sites by Native Chemical Modification

The ability to obtain a homogeneous sample of protein is invaluable when studying the effect of alterations such as post-translational modifications (PTMs). Selective functionalization of a protein to investigate the effect of PTMs on its structure or activity can be achieved by chemical modification of cysteine residues. We demonstrate here that one such technique, which involves conversion of cysteine to dehydroalanine followed by thiol nucleophile addition, is suitable for the site-specific installation of a wide range of chemical mimics of PTMs, including acetylated and dimethylated lysine, and other unnatural amino acids. These reactions, optimized for the clinically relevant kinase Aurora-A, readily proceed to completion as revealed by intact protein mass spectrometry. Moreover, these reactions proceed under non-denaturing conditions, which is desirable when working with large protein substrates. We have determined reactivity trends for a diverse range of thiol nucleophile addition reactions at two separate sites on Aurora-A, and we also highlight limitations when using thiol nucleophiles that contain basic functional groups. We show that chemical modification of cysteine residues is possible not only on a flexible surface-exposed loop, but also within a deep active site pocket at the conserved DFG motif, which reveals the potential use of this method in exploring enzyme function through modification of catalytic site residues.     

Role of cysteine residues in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase. Site-directed mutagenesis and characterization of the mutant enzymes

Each of the four identical subunits of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase contains two cysteine residues, Cys156 and Cys296 (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001). Both are accessible to modification by sulfhydryl reagents under nondenaturing conditions (Jordan-Starck, T. C., and Rodwell, V. W. (1989) J. Biol. Chem. 264, 17913-17918). We used site-directed mutagenesis to construct three mutant enzymes in which alanine replaced either or both cysteine residues. Mutant enzymes C156A, C296A, and C156/296A were over-expressed in Escherichia coli and were found to be fully active. Following their purification, all four forms of the enzyme were compared with respect to their catalytic efficiency, their affinities for the substrates of all four catalyzed reactions, and for their sensitivity to inactivation by sulfhydryl reagents. Replacement of cysteine residues with alanine residues had no major effect on either the specific activity or the affinity of the enzymes for any substrate. The mutants catalyzed all four HMG-CoA reductase reactions as efficiently as did the wild-type enzyme, and coenzyme A stimulated mevaldehyde reduction to the same extent as for wild-type HMG-CoA reductase. Mutant C156A and the cysteine-free mutant C156/296A were not inactivated by 5,5'-dithiobis(2-nitrobenzoate). By contrast, mutant C296A was inactivated to the same extent as was the wild-type enzyme. Following treatment of the mutant enzymes with N-ethylmaleimide, the four reductase reactions catalyzed by mutant C296A were inactivated to the same extent as for the wild-type enzyme. Neither mutant C156A nor C156/296A was affected by this reagent. We conclude that the sulfhydryl reagent-reactive group whose derivatization leads to loss of enzymatic activity is Cys156. However, this residue is not an essential active site residue since neither substrate binding nor catalysis was affected when it was replaced by alanine. Possible roles of cysteine in maintaining structural stability are discussed.     

Chemical modification of chalcone isomerase by diethyl pyrocarbonate: histidine residues are not essential for catalysis

Chalcone isomerase form soybean is inactivated by treatment with diethyl pyrocarbonate (DEP). The competitive inhibitor 4',4-dihydroxychalcone provides kinetic protection against inactivation by DEP with a binding constant at the site of protection in agreement with its binding constant at the active site. Very high concentrations of the competitive inhibitors 4',4-dihydroxychalcone or morin hydrate offer a 10- to 40-fold maximal protection, suggesting a second slower mechanism for inactivation which cannot be prevented by blockage of the active site. Blockage of the only cysteine residue in chalcone isomerase with p-mercuribenzoate does not affect the rate constant for DEP-dependent inactivation and indicates that the modification of the cysteine residue is not responsible for the activity loss observed in the presence of DEP. Treatment of inactivated enzyme with hydroxylamine does not restore catalytic activity, indicating that the modification of histidine or tyrosine residues is not responsible for the activity loss. All five histidines of chalcone isomerase are modified by DEP at pH 5.7 and ionic strength 1.0 M. The rate constant for the modification of the histidine residues of chalcone isomerase is close to that for the reaction of N-acetyl histidine with DEP, indicating that the histidine residues are quite accessible to the modifying reagent. The rate of histidine modification is the same in native enzyme, in urea-denatured enzyme, and in the presence of a competitive inhibitor. In the presence of the competitive inhibitor morin hydrate, all of the histidine residues of chalcone isomerase can be modified without significant loss in catalytic activity. These results demonstrate that the histidine residues of chalcone isomerase are not essential for catalysis and therefore cannot function as nucleophilic catalysts as previously proposed.     

Structure and mechanism of peptide methionine sulfoxide reductase, an "anti-oxidation" enzyme

Peptide methionine sulfoxide reductase (MsrA) reverses oxidative damage to both free methionine and methionine within proteins. As such, it helps protect the host organism against stochastic damage that can contribute to cell death. The structure of bovine MsrA has been determined in two different modifications, both of which provide different insights into the biology of the protein. There are three cysteine residues located in the vicinity of the active site. Conformational changes in a glycine-rich C-terminal tail appear to allow all three thiols to come together and to participate in catalysis. The structures support a unique, thiol-disulfide exchange mechanism that relies upon an essential cysteine as a nucleophile and additional conserved residues that interact with the oxygen atom of the sulfoxide moiety.     

Purification and characterization of ACR2p, the Saccharomyces cerevisiae arsenate reductase

In Saccharomyces cerevisiae, expression of the ACR2 and ACR3 genes confers arsenical resistance. Acr2p is the first identified eukaryotic arsenate reductase. It reduces arsenate to arsenite, which is then extruded from cells by Acr3p. In this study, we demonstrate that ACR2 complemented the arsenate-sensitive phenotype of an arsC deletion in Escherichia coli. ACR2 was cloned into a bacterial expression vector and expressed in E. coli as a C-terminally histidine-tagged protein that was purified by sequential metal chelate affinity and gel filtration chromatography. Acr2p purified as a homodimer of 34 kDa. The purified protein was shown to catalyze the reduction of arsenate to arsenite. Enzymatic activity as a function of arsenate concentration exhibited an apparent positive cooperativity with an apparent Hill coefficient of 2.7. Activity required GSH and glutaredoxin as the source of reducing equivalents. Thioredoxin was unable to support arsenate reduction. However, glutaredoxins from both S. cerevisiae and E. coli were able to serve as reductants. Analysis of grx mutants lacking one or both cysteine residues in the Cys-Pro-Tyr-Cys active site demonstrated that only the N-terminal cysteine residue is essential for arsenate reductase activity. This suggests that during the catalytic cycle, Acr2p forms a mixed disulfide with GSH before being reduced by glutaredoxin to regenerate the active Acr2p reductase.     

The role of thioredoxin in filamentous phage assembly. Construction, isolation, and characterization of mutant thioredoxins

Filamentous phage assembly in vivo shows an absolute requirement for thioredoxin and a partial requirement for thioredoxin reductase. Mutants in which one or both of the active site cysteine residues of thioredoxin were changed to alanine or serine were constructed and shown to support filamentous phage assembly. Some of the mutants were almost as effective as wild-type thioredoxin, while others supported phage assembly only when high levels of the mutant protein were present in the infected cell. The mutant proteins were all inactive in an assay which couples oxidation of NADPH to reduction of 5,5'-dithiobis-2-nitrobenzoic acid) via thioredoxin reductase and thioredoxin. These active site mutants make phage assembly completely independent of thioredoxin reductase, which suggests that the phage needs, and the active site mutants provide, the proteins in the reduced conformation. Other mutants were isolated on the basis of their failure to support filamentous phage growth. These specified mutant thioredoxin proteins with varying levels of redox activity in vivo and in vitro. The locations of these mutations suggest that the surface of thioredoxin thought to interact with thioredoxin reductase also interacts with the filamentous phage assembly machinery. An in vivo assay for thioredoxin redox function, based on the ability of cells to utilize methionine sulfoxide, was developed. Met- cells containing mutant thioredoxins that are inactive in vitro do not form colonies on plates containing methionine sulfoxide as the sole methionine source.     

Re-evaluation of the role of thiol groups in rabbit muscle aldolase A

Exposed thiol groups of rabbit muscle aldolase A were modified by 5,5'-dithiobis(2-nitrobenzoic) acid with concomittant loss of enzyme activity. When 5-thio-2-nitrobenzoate residues bound to enzyme SH groups were replaced by small and uncharged cyanide residues the enzyme activity was restored by more than 50%. The removal of a bulky C-terminal tyrosine residue from the active site of aldolase A resulted in enzyme which was inhibited by 5,5'-dithiobis(2-nitrobenzoic) acid only by 50% and its activity was nearly unchanged after modification of its thiol groups with cyanide. The results obtained show directly that rabbit muscle aldolase A does not possess functional cysteine residues and that the inactivation of the enzyme caused by sulfhydryl group modification reported previously can be attributed most likely to steric hindrance of a catalytic site by modifying agents.     

The role of zinc with special reference to the essential thiol groups in delta-aminolevulinic acid dehydratase of bovine liver.

delta-Aminolevulinic acid dehydratase (5-aminolevulinic acid hydro-lyase (adding 5-aminolevulinic acid and cyclizing), EC 4.2.1.24 purified from bovine liver in the presence of both SH-reducing reagent and zinc during the purification contained one zinc atom and eight SH groups/subunit. This preparation showed the full enzymatic activity even in the absence of thiol activator. It was found that two cysteine residues, one zinc atom and two histidine residues were involved in the active site. The enzyme was fullly active as long as two SH groups in the active site remained in the reduced form even in the absence of zinc. However, the enzymatic activity was completely lost, with a concomitant loss of bound zinc, upon oxidation of the SH groups to a disulfide bond, modification of SH groups with chemical reagents, or mercaptide formation by heavy metals. Thus, it is apparent that the activity depends on the essential SH groups. The zinc is not absolutely essential for the activity but may be required to prevent the essential SH groups from autooxidation by coordination. Binding experiments indicated that there was one binding site of zinc/subunit. Photooxidation of histidine residues diminished both enzymatic activity and bound zinc, suggesting that the histidine residues not only constituted the active site but also served as a possible ligand to zinc.     

Selective inactivation of glutaredoxin by sporidesmin and other epidithiopiperazinediones

Glutaredoxin (thioltransferase) is a thiol-disulfide oxidoreductase that displays efficient and specific catalysis of protein-SSG deglutathionylation and is thereby implicated in homeostatic regulation of the thiol-disulfide status of cellular proteins. Sporidesmin is an epidithiopiperazine-2,5-dione (ETP) fungal toxin that disrupts cellular functions likely via oxidative alteration of cysteine residues on key proteins. In the current study sporidesmin inactivated human glutaredoxin in a time- and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductase enzymes, glutathione reductase, thioredoxin, and thioredoxin reductase, were unaffected by sporidesmin. Inactivation of glutaredoxin required the reduced (dithiol) form of the enzyme, the oxidized (intramolecular disulfide) form of sporidesmin, and molecular oxygen. The inactivated glutaredoxin could be reactivated by dithiothreitol only in the presence of urea, followed by removal of the denaturant, indicating that inactivation of the enzyme involves a conformationally inaccessible disulfide bond(s). Various cysteine-to-serine mutants of glutaredoxin were resistant to inactivation by sporidesmin, suggesting that the inactivation reaction specifically involves at least two of the five cysteine residues in human glutaredoxin. The relative ability of various epidithiopiperazine-2,5-diones to inactivate glutaredoxin indicated that at least one phenyl substituent was required in addition to the epidithiodioxopiperazine moiety for inhibitory activity. Mass spectrometry of the modified protein is consistent with formation of intermolecular disulfides, containing one adducted toxin per glutaredoxin but with elimination of two sulfur atoms from the detected product. We suggest that the initial reaction is between the toxin sulfurs and cysteine 22 in the glutaredoxin active site. This study implicates selective modification of sulfhydryls of target proteins in some of the cytotoxic effects of the ETP fungal toxins and their synthetic analogues.     

Conformation and microenvironment of the active site of a low molecular weight 1,4-beta-D-glucan glucanohydrolase from an alkalothermophilic Thermomonospora sp.: involvement of lysine and cysteine residues

Conformation and microenvironment at the active site of 1,4-beta-D-glucan glucanohydrolase was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde. OPTA has been known to form a fluorescent isoindole derivative by cross-linking the proximal thiol and amino groups of cysteine and lysine. Modification of lysine of the enzyme by TNBS and of cysteine residue by PHMB abolished the ability of the enzyme to form an isoindole derivative with OPTA. Kinetic analysis of the TNBS and PHMB-modified enzyme suggested the presence of essential lysine and cysteine residues, respectively, at the active site of the enzyme. The substrate protection of the enzyme with carboxymethylcellulose (CMC) confirmed the involvement of lysine and cysteine residues in the active site of the enzyme. Multiple sequence alignment of peptides obtained by tryptic digestion of the enzyme showed cysteine is one of the conserved amino acids corroborating the chemical modification studies.     

Amino acid sequence of COOH-terminal 20K Da fragment from pig liver microsomal NADPH-cytochrome P-450 reductase

We have determined the complete amino acid sequence of a 20K Da COOH-terminal fragment of porcine NADPH-cytochrome P-450 reductase. The 20K Da fragment is probably produced by a proteolytic cleavage of the intact protein in porcine liver microsomes, and since the cleavage does not affect enzymatic activity, the fragment has been studied as a distinct domain. The sequence comprises 175 amino acids including three cysteine residues, one of which has been previously identified as protected by NADPH from S-carboxymethylation. The NADPH-protected cysteine lies in a stretch of 12 residues with partial homology to glutathione reductase, and is adjacent to a hydrophobic region containing a glycine-rich stretch homologous to other FAD-containing proteins. The predicted secondary structure over this entire region is beta-sheet/beta-turn/beta-sheet/alpha-helix/beta-sheet/beta-turn/alpha-h elix corresponding to hydrophobic residues 21-28/glycine-rich residues 29-33/residues 34-38/residues 39-54/residues 56-61/NADPH-protected cysteine residues 62-78/residues 71-82. It is possible that the 20K Da domain provided a significant portion of the sequence responsible for binding FAD and NADPH in the intact enzyme. This data provides a basis for further active site studies.