Biosynthesis of the iron-molybdenum cofactor of nitrogenase.

The iron-molybdenum cofactor (the M-cluster) serves as the active site of molybdenum nitrogenase. Arguably one of the most complex metal cofactors in biological systems, the M-cluster is assembled through the formation of an 8Fe core prior to the insertion of molybdenum and homocitrate into this core. Here, we review the recent progress in the research area of M-cluster assembly, with an emphasis on our work that provides useful insights into the mechanistic details of this process.

300-Fold increase in production of the Zn2+-dependent dechlorinase TrzN in soluble form via apoenzyme stabilization.

Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.

Activation of HydA(DeltaEFG) requires a preformed [4Fe-4S] cluster.

The H-cluster is a complex bridged metal assembly at the active site of [FeFe]-hydrogenases that consists of a [4Fe-4S] subcluster bridged to a 2Fe-containing subcluster with unique nonprotein ligands, including carbon monoxide, cyanide, and a dithiolate ligand of unknown composition. Specific biosynthetic gene products (HydE, HydF, and HydG) responsible for the biosynthesis of the H-cluster and the maturation of active [FeFe]-hydrogenase have previously been identified and shown to be required for the heterologous expression of active [FeFe]-hydrogenase [Posewitz, M. C., et al. (2004) J. Biol. Chem. 279, 25711-25720]. The precise roles of the maturation proteins are unknown; the most likely possibility is that they are directed at the synthesis of the entire 6Fe-containing H-cluster, the 2Fe subcluster, or only the unique ligands of the 2Fe subcluster. The spectroscopic and biochemical characterization of HydA(DeltaEFG) (the [FeFe]-hydrogenase structural protein expressed in the absence of the maturation machinery) reported here indicates that a [4Fe-4S] cluster is incorporated into the H-cluster site. The purified protein in a representative preparation contains Fe (3.1 +/- 0.5 Fe atoms per HydA(DeltaEFG)) and S(2-) (1.8 +/- 0.5 S(2-) atoms per HydA(DeltaEFG)) and exhibits UV-visible spectroscopic features characteristic of iron-sulfur clusters, including a bleaching of the visible chromophore upon addition of dithionite. The reduced protein gave rise to an axial S = (1)/(2) EPR signal (g = 2.04 and 1.91) characteristic of a reduced [4Fe-4S](+) cluster. Mossbauer spectroscopic characterization of (57)Fe-enriched HydA(DeltaEFG) provided further evidence of the presence of a redox active [4Fe-4S](2+/+) cluster. Iron K-edge EXAFS data provided yet further support for the presence of a [4Fe-4S] cluster in HydA(DeltaEFG). These spectroscopic studies were combined with in vitro activation studies that demonstrate that HydA(DeltaEFG) can be activated by the specific maturases only when a [4Fe-4S] cluster is present in the protein. In sum, this work supports a model in which the role of the maturation machinery is to synthesize and insert the 2Fe subcluster and/or its ligands and not the entire 6Fe-containing H-cluster bridged assembly.

Role of UME6 in transcriptional regulation of a DNA repair gene in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae UV radiation and a variety of chemical DNA-damaging agents induce the transcription of specific genes, including several involved in DNA repair. One of the best characterized of these genes is PHR1, which encodes the apoenzyme for DNA photolyase. Basal-level and damage-induced expression of PHR1 require an upstream activation sequence, UAS(PHR1), which has homology with DRC elements found upstream of at least 19 other DNA repair and DNA metabolism genes in yeast. Here we report the identification of the UME6 gene of S. cerevisiae as a regulator of UAS(PHR1) activity. Multiple copies of UME6 stimulate expression from UAS(PHR1) and the intact PHR1 gene. Surprisingly, the effect of deletion of UME6 is growth phase dependent. In wild-type cells PHR1 is induced in late exponential phase, concomitant with the initiation of glycogen accumulation that precedes the diauxic shift. Deletion of UME6 abolishes this induction, decreases the steady-state concentration of photolyase molecules and PHR1 mRNA, and increases the UV sensitivity of a rad2 mutant. Despite the fact that UAS(PHR1) does not contain the URS1 sequence, which has been previously implicated in UME6-mediated transcriptional regulation, we find that Ume6p binds to UAS(PHR1) with an affinity and a specificity similar to those seen for a URS1 site. Similar binding is also seen for DRC elements from RAD2, RAD7, and RAD53, suggesting that UME6 contributes to the regulated expression of a subset of damage-responsive genes in yeast.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase from Pyrococcus horikoshii.

Galactokinase (EC 2.7.1.6) catalyzes the ATP-dependent phosphorylation of alpha-D-galactose to alpha-D-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with alpha-D-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 A resolution for the apo form and to 1.7 A for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 A, beta = 109.8 degrees. The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.

Synthesis of cytochrome c oxidase in the liver of Rana catesbeiana tadpole treated with griseofulvin.

The effect of griseofulvin treatment on the synthesis of cytochrome c oxidase was studied with the liver of the tadpole, Rana catesbeiana. (1) In the liver of tadpole treated with griseofulvin, a ferrochelatase inhibitor, the synthesis of heme a, but not cytochrome c oxidase protein, is inhibited. (2) The apocytochrome c oxidase which is formed in the liver of tadpole treated with griseofulvin is converted to the active holoenzyme by exogenously added heme a.

Purification of beef kidney D-aspartate oxidase overexpressed in Escherichia coli and characterization of its redox potentials and oxidative activity towards agonists and antagonists of excitatory amino acid receptors.

The flavoenzyme d-aspartate oxidase from beef kidney (DASPO, EC 1.4. 3.1) has been overexpressed in Escherichia coli. A purification procedure, faster than the one used for the enzyme from the natural source (bDASPO), has been set up yielding about 2 mg of pure recombinant protein (rDASPO) per each gram of wet E. coli paste. rDASPO has been shown to possess the same general biochemical properties of bDASPO, except that the former contains only FAD, while the latter is a mixture of two forms, one active containing FAD and one inactive containing 6-OH-FAD (9-20% depending on the preparation). This results in a slightly higher specific activity (about 15%) for rDASPO compared to bDASPO and in facilitated procedures for apoprotein preparation and reconstitution. Redox potentials of -97 mV and -157 mV were determined for free and l-(+)-tartrate complexed DASPO, respectively, in 0.1 M KPi, pH 7.0, 25 degrees C. The large positive shift in the redox potential of the coenzyme compared to free FAD (-207 mV) is in agreement with similar results obtained with other flavooxidases. rDASPO has been used to assess a possible oxidative activity of the enzyme towards a number of compounds used as agonists or antagonists of neurotransmitters, including d-aspartatic acid, d-glutamic acid, N-methyl-d-aspartic acid, d,l-cysteic acid, d-homocysteic acid, d, l-2-amino-3-phosphonopropanoic acid, d-alpha-aminoadipic acid, d-aspartic acid-beta-hydroxamate, glycyl-d-aspartic acid and cis-2, 3-piperidine dicarboxylic acid. Kinetic parameters for each substrate in 50 mM KPi, pH 7.4, 25 degrees C are reported.

Expression of prokaryotic and eukaryotic cytochromes c in Escherichia coli.

C-type cytochromes from various sources show substantial structural conservation. For the covalent attachment of heme groups to apocytochromes, however, three different enzyme systems have been described so far. We have examined the ability of the heme ligation systems of Escherichia coli and of Saccharomyces cerevisiae to process cytochromes from S. cerevisiae, Paracoccus denitrificans, and Synechocystis sp. PCC 6803. E. coli's maturation system with at least eight different proteins accepted all these cytochromes for heme ligation. The single subunit heme lyase from S. cerevisiae mitochondria, on the other hand, failed to attach heme groups to cytochromes of prokaryotic origin.

Heme maintains catalytically active structure of cytochrome P-450.

Treatment of purified cytochrome P-450 LM2 and its liposome-bound form with hydrogen peroxide led to complete destruction of the P-450 heme. The apoenzyme thus produced could be reconstituted to catalytically active cytochrome P-450 by incubation with hemin, the reconstitution efficiency being 50% for the soluble enzyme and 80% for the liposome-bound enzyme. The removal of heme from the soluble hemoprotein resulted in a 3-fold decrease in the efficiency of its incorporation into sonicated liposomes. The contents of 5 secondary structure forms in the native, apo- and reconstituted holoenzymes were estimated from their circular dichroism spectra. It was thus found that the helix content increased from 34% to 60% upon removal of the heme from the native enzyme. We suggest that the increase in the helix content leads to a reduction of the incorporation efficiency into liposomal membranes.

Differential sensitivity of rat hepatocyte CYP isoforms to self-generated nitric oxide.

Early loss of P450 in rat hepatocyte cultures appears directly related to nitric oxide (NO) overproduction. This study investigates the influence of endogenously generated NO (or NO-derived species) on the relative expression of cytochrome P450 (CYP) isoforms in rat hepatocytes. Our results support the view that loss of P450 holoenzyme in culture is the ultimate consequence of a NO driven process, activated during the common hepatocyte isolation procedure, that leads to an accelerated and selective degradation of specific CYP apoproteins. Under conditions in which NO and peroxynitrite formation is operative, changes in the level of specific CYP isoforms result in a significant alteration of the CYP apoprotein profile that after 24 h of culture is quite different from that found in the liver of uninduced rats. This process is reverted by the early and efficient inhibition of NO synthesis, which allows for (1) maintenance of total P450 holoenzyme content, (2) preservation of the initial constitutive CYP pattern in culture and (3) the early expression of the normal inducibility in response to model inducers.

Expression and distribution of cytochrome P450 enzymes in male rat kidney: effects of ethanol, acetone and dietary conditions.

Ethanol, acetone, diet and starvation, known modulators of the hepatic cytochrome P450 (CYP)-dependent microsomal monooxygenase system, were assessed for their effects on cytochrome P450 isozyme content and monooxygenase activities in the male rat kidney. In acute experiments, rats were either treated with acetone, fasted or given a combination of the two treatments. Acetone treatment alone induced CYP2E1-dependent p-nitrophenol hydroxylase activity in kidney microsomes by 8-fold. This was accompanied by a 6-fold increase in CYP2E1 apoprotein as determined by Western blot analysis. There was, however, no significant increase in steady-state levels of CYP2E1 mRNA as measured by Northern blot analysis. Starvation also induced CYP2E1 apoprotein in the kidney and, as has been reported previously in the liver, had a synergistic inductive effect with acetone. CYP2B and CYP3A apoproteins were also induced by acetone, starvation and starvation/acetone combinations in the kidney. Immunohistochemical analysis revealed localization of CYP2E1 and CYP2B principally in the cortex associated with tubular cells. This distribution was maintained upon starvation/acetone treatment. Two induction experiments were performed in which the ethanol was administered as part of a system of total enteral nutrition (TEN). A short-term study was conducted in which ethanol was administered for 8 days in two liquid diets of different composition, and a chronic experiment was performed in which ethanol was administered for 35 days. A diet-independent 6-fold increase in CYP2E1 apoprotein was observed in the short-term experiment. Expression of CYP3A and CYP2A cross-reactive apoproteins in kidney microsomes appeared to be affected by alterations in diet but, were unaffected by ethanol treatment. In the chronic 35-day ethanol exposure experiment, CYP2E1 apoprotein was also elevated 6-fold and this was found to be accompanied by a significant 3-fold increase in CYP2E1 mRNA. In the same study, no ethanol effects were apparent on expression of CYP2B and CYP3A apoproteins. Thus, acetone induced a variety of renal cytochrome P450 forms in addition to CYP2E1, while ethanol appeared to be a much more specific renal CYP2E1 inducer. Furthermore, as reported in the liver, acetone and ethanol appeared to induce CYP2E1 in the kidney by different mechanisms.

Characterization of cytochrome P450 2E1 induction in a rat hepatoma FGC-4 cell model by ethanol.

The hepatic microsomal ethanol-oxidizing system (MEOS) has been well characterized as an important pathway in ethanol metabolism. Cytochrome P450 2E1 (CYP 2E1), the principal component of MEOS, is ethanol inducible and has been implicated in hepatotoxicity associated with alcohol abuse and exposure to organic solvents. Results of chronic in vivo experiments have shown that ethanol induction of hepatic CYP 2E1 occurs by a two-step mechanism. The first step of induction is associated with low blood alcohol concentrations (BACs) and appears to be post-transcriptional, whereas high BACs observed in step-two induction are associated with increased CYP 2E1 gene transcription. The mechanisms underlying these induction steps are under intense investigation. Progress in this area has been limited due to lack of hepatic cell culture models that express CYP 2E1. We report here an in vitro tissue culture cell model, the FGC-4 hepatoma cell line, that exhibits basal levels of CYP 2E1 apoprotein that are inducible by ethanol treatment. Total cellular RNA and microsomal fractions were isolated from control or ethanol-treated confluent cells, and CYP 2E1 mRNA and apoprotein levels were characterized by northern blot or immunoblot analysis, respectively. Initial experiments on isolated microsomes revealed detectable levels of CYP 2E1 apoprotein in control cells that were induced 5-fold in cells treated with 100 mM ethanol for 24 hr. Concentration-response experiments demonstrated that the maximal 24-hr induction in CYP 2E1 apoprotein level was 5-fold and was attained at a concentration of 10 mM ethanol. Interestingly, while the steady-state mRNA levels encoding CYP 2E1 were detectable, they remained unchanged in identically treated cells. Furthermore, there was no observed increase in CYP 2E1 mRNA levels in an extended time course to 72 hr or at higher alcohol concentrations (up to 1500 mM), providing preliminary evidence that the induction is post-transcriptional. The time course of CYP 2E1 apoprotein induction by exposure to 100 mM ethanol demonstrated maximal induction at 8 hr. Measurement of CYP 2E1 apoprotein levels after removal of ethanol from pretreated cells demonstrated the half-life of the apoprotein to be 12.7 hr, in good agreement with previous reports using primary hepatocytes. The half-life of the induced protein after ethanol removal in the presence of cyclohexamide (10 micrograms/mL) was biphasic with a rapid 1.8 hr first phase followed by a slower 44.7 hr second phase.(ABSTRACT TRUNCATED AT 400 WORDS)

Biosynthesis of covalently bound flavin: isolation and in vitro flavinylation of the monomeric sarcosine oxidase apoprotein.

The covalently bound FAD in native monomeric sarcosine oxidase (MSOX) is attached to the protein by a thioether bond between the 8alpha-methyl group of the flavin and Cys315. Large amounts of soluble apoenzyme are produced by controlled expression in a riboflavin-dependent Escherichia coli strain. A time-dependent increase in catalytic activity is observed upon incubation of apoMSOX with FAD, accompanied by the covalent incorporation of FAD to approximately 80% of the level observed with the native enzyme. The spectral and catalytic properties of the reconstituted enzyme are otherwise indistinguishable from those of native MSOX. The reconstitution reaction exhibits apparent second-order kinetics (k = 139 M(-)(1) min(-)(1) at 23 degrees C) and is accompanied by the formation of a stoichiometric amount of hydrogen peroxide. A time-dependent reduction of FAD is observed when the reconstitution reaction is conducted under anaerobic conditions. The results provide definitive evidence for autoflavinylation in a reaction that proceeds via a reduced flavin intermediate and requires only apoMSOX and FAD. Flavinylation of apoMSOX is not observed with 5-deazaFAD or 1-deazaFAD, an outcome attributed to a decrease in the acidity of the 8alpha-methyl group protons. Covalent flavin attachment is observed with 8-nor-8-chloroFAD in an aromatic nucleophilic displacement reaction that proceeds via a quininoid intermediate but not a reduced flavin intermediate. The reconstituted enzyme contains a modified cysteine-flavin linkage (8-nor-8-S-cysteinyl) as compared with native MSOX (8alpha-S-cysteinyl), a difference that may account for its approximately 10-fold lower catalytic activity.

Integration of the thylakoid membrane protein cytochrome b6 in the cytoplasmic membrane of Escherichia coli.

An overexpression system for spinach apocytochrome b(6) as a fusion protein to a maltose-binding protein in Escherichia coli was established using the expression vector pMalp2. The fusion of the cytochrome b(6) to the periplasmic maltose-binding protein directs the cytochrome on the Sec-dependent pathway. The cytochrome b(6) has a native structure in the bacterial cytoplasmic membrane with both NH(2) and COOH termini on the same, periplasmic side of the membrane but has the opposite orientation compared to that in thylakoid. Our data also show that in the E. coli cytoplasmic membrane, apocytochrome b(6) and exogenic hemes added into a culture media spontaneously form a complex with similar spectroscopic properties to native cytochrome b(6). Reconstituted membrane-bound cytochrome b(6) contain two b hemes (alpha band, 563 nm; average E(m,7) = -61 +/- 0.84 and -171 +/- 1.27 mV).

Production, characterization, and reconstitution of recombinant quinoprotein glucose dehydrogenase (soluble type; EC 1.1.99.17) apoenzyme of Acinetobacter calcoaceticus.

Soluble, periplasmic quinoprotein glucose dehydrogenase of Acinetobacter calcoaceticus (sGDH; EC 1.1.99.17) was produced in good yield in the apoenzyme form (without the cofactor pyrroloquinoline quinone, PQQ) by an Escherichia coli recombinant strain provided with a plasmid containing the gene under control of a lac promoter. Structural analysis of the purified apoenzyme revealed that the E. coli strain used produces the correct mature protein. Titration of the apoenzyme with PQQ in the presence of Ca2+ showed that a linear relation exists between the amount of added PQQ and activity observed, and that the subunit and PQQ associate in a molar ratio of 1:1. Based on spectral and enzymatic criteria, it is concluded that the present holoenzyme preparation has a better quality than the previously described preparations of authentic holoenzyme. As isolated here, the recombinant apoenzyme was in the dimeric form. Partial monomerization occurred upon gel filtration in a buffer with chelator and the process could be reversed with Ca2+. PQQ binds to the dimer in the presence of chelator, not to the monomer. However, the PQQ-containing dimer was not active and showed an unusual absorption spectrum which was slowly converted into a PQQH2-like spectrum when glucose was added. Full restoration of activity was achieved upon addition of Ca2+ and the spectra were immediately converted into those of normal holoenzyme in the oxidized and reduced form, respectively. Addition of chelator to holoenzyme did not lead to inactivation or monomerization. It is concluded, therefore, that Ca2+ has a dual role in this enzyme, being required for dimerization of the subunits as well as for functionalization of the bound PQQ, and that it is more firmly attached to the holoenzyme than to the apoenzyme.

The relation of heme to catalase apoprotein synthesis in yeast.

The synthesis of two hemoproteins, catalase A and catalase T, was studied in mutants of Saccharomyces cerevisiae deficient in heme formation. These mutants can be grown on the end-product heme or on a heme precursor, or on ergosterol and Tween 80 (a source of oleic acid). It was found by immunoprecipitation that, in the presence of heme, catalases A and T were present in the mutants, but that in its absence (growth on ergosterol and Tween 80) the apoproteins of these enzymes were not detectable. In contrast, cytochrome c peroxidase, and some of the subunits of cytochrome c oxidase are present in cells grown without heme (Saltzgaber-Müller, J., and Schatz, G. (1978) J. Biol. Chem. 253, 305-310). Other evidence suggests that absence of catalase T apoprotein under heme-less conditions may be due to control by heme of apoprotein synthesis (G. Ammerer and H. Ruis, unpublished results), rather than increased proteolytic degradation.

Biogenesis of cytochrome c in Neurospora crassa. Synthesis of apocytochrome c, transfer to mitochondria and conversion to Holocytochrome c.

1. Precipitating antibodies specific for apocytochrome c and holocytochrome c, respectively, were employed to study synthesis and intracellular transport of cytochrome c in Neurospora in vitro. 2. Apocytochrome c as well as holocytochrome c were found to be synthesized in a cell-free homogenate. A precursor product relationship between the two components is suggested by kinetic experiments. 3. Apocytochrome c synthesized in vitro was found in the post-ribosomal fraction and not in the mitochondrial fraction, whereas holocytochrome c synthesized in vitro was mainly detected in the mitochondrial fraction. A precursor product relationship between postribosomal apocytochrome c and mitochondrial holocytochrome c is indicated by the labelling data. In the microsomal fraction both apocytochrome c and holocytochrome c were found in low amounts. Their labeling kinetics do not subbest a precursor role of microsomal apocytochrome c or holocytochrome c. 4. Formation of holocytochrome c from apocytochrome c was observed when postribosomal supernatant containing apocytochrome c synthesized in vitro was incubated with isolated mitochondria, but not when incubated in the absence of mitochondria. The cytochrome c formed under these conditions was detected in the mitochondria. 5. Conversion of labelled apocytochrome c synthesized in vitro to holocytochrome c during incubation of a postribosomal supernatant with isolated mitochondria was inhibited when excess isolated apocytochrome c, but not when holocytochrome c was added. 6. The data presented are interpreted to show that apocytochrome c is synthesized on cytoplasmic ribosomes and released into the supernatant. It is suggested that apocytochrome c migrates to the inner mitochondrial membrane, where the heme group is covalently linked to the apoprotein. The hypothesis is put forward that the concomitant change in conformation leads to trapping of holocytochrome c in the membrane. The problems of permeability of the outer mitochondrial membrane to apocytochrome c and the site and nature of the reaction by which the heme group is linked to the apoprotein are discussed.

Reactions of phosphonate analogs of pyridoxal phosphate with apo-aspartate aminotransferase.

We have investigated reactions of the 5-phosphonoethyl and 5-phosphonoethenyl analogs of pyridoxal 5'-phosphate in the coenzyme site of cytosolic aspartate aminotransferase. Acid dissociation constants and equilibrium constants for hydration and for tautomerization have been evaluated for these compounds. In confirmation of previous results, both compounds are partially active. They bind to apoenzyme well and undergo conversion in the presence of glutamate to amine forms which show induced circular dichroism comparable to that of native enzyme. A normal "external" Schiff base is evidently formed with 2-methylaspartate, but the amounts of quinonoid intermediate formed with erythro-3-hydroxyaspartate are less than those formed with pyridoxal phosphate. The pKa of the imine group of the enzyme reconstituted with the phosphonoethyl analog is more than two units lower than that in the native enzyme. Binding of the dicarboxylates glutarate, 2-oxoglutarate, and succinate shifts the pKa upward. The absorption spectra of the resulting complexes indicate the existence of at least three low pH species. A shift of 2.3 to 2.9 ppm to a lower frequency was observed for the 31P NMR signal upon binding of these dicarboxylates or of 2-methylaspartate. Enzyme containing the analogs crystallizes. Polarized absorption spectra suggest that the coenzyme has an orientation similar to that of pyridoxal phosphate in the native enzyme.

Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli production of soluble apoenzyme and in vitro formation of active holoenzyme.

Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli BL21(DE3) was accomplished by introducing a derivative of pET-23a(+) containing a copy of the coding gene into the multicloning site. E. coli BL21(DE3)/pETMNCAT produced abundant quantities of manganese catalase as insoluble inclusion bodies. Regeneration of active catalase was achieved by denaturation in guanidine hydrochloride and subsequent dialysis in the presence of manganese ion. When the E. coli chaperone genes GroEL, GroES, DnaK, DnaJ and GrpE were coexpressed with manganese catalase, a significant fraction of the overproduced protein was partitioned into the soluble fraction. However, almost all of the soluble enzyme was isolated in a manganese-deficient apo form which could subsequently be converted into active holoenzyme by incubation with manganese ion at high temperatures. Further experiments on this apo catalase suggested that the structure of this protein was virtually identical to the active holoenzyme.