Peter C. Loewen

Diversity of structures and properties among catalases

More than 300 catalase sequences are now available, divided among monofunctional catalases (> 225), bifunctional catalase-peroxidases (> 50) and manganese-containing catalases (> 25). When combined with the recent appearance of crystal structures from at least two representatives from each of these groups (nine from the monofunctional catalases), valuable insights into the catalatic reaction mechanism in its various forms and into catalase evolution have been gained. The structures have revealed an unusually large number of modifications unique to catalases, a result of interacting with reactive oxygen species. Biochemical and physiological characterization of catalases from many different organisms has revealed a surprisingly wide range of catalatic efficiencies, despite similar sequences. Catalase gene expression in micro-organisms generally is controlled either by sensors of reactive oxygen species or by growth phase regulons, although the detailed mechanisms vary considerably.

Enzymology and structure of catalases

Abstract Catalases continue to present a challenge and are an object of interest to the biochemist despite more than 100 years of study. More than 120 sequences, seven crystal structures, and a wealth of kinetic and physiological data are currently available, from which considerable insight into the catalytic mechanism has been gained. Indeed, even the crystal structures of some of the presumed reaction intermediates are available. This body of information continues to accumulate almost daily. Have we exhausted catalases as a source of information about protein structure and the catalatic mechanisms? The answer is clearly no. With each structure reported comes new information, often including structural modifications seemingly unique to catalases and with roles that remain to be explained. Despite a deeply buried active site, catalases exhibit one of the fastest turnover rates determined. This presents the as yet unanswered question of how substrate can access the active site while products are simultaneously exhausted with a potential turnover rate of up to 10 6 per second. The complex folding pathway that produces the intricate interwoven arrangement of subunits also remains to be fully clarified.

Catalases HPI and HPII in Escherichia coli are induced independently

Three strains of Escherichia coli differing only in the catalase locus mutated by transposon Tn10 were constructed. These strains produced only catalase HPI (katE::Tn10 and katF::Tn10 strains) or catalase HPII (katG::Tn10). HPI levels increased gradually about twofold during logarithmic growth but did not increase during growth into stationary phase in rich medium. HPII levels, which were initially threefold lower than HPI levels, did not change during logarithmic growth but did increase tenfold during growth into stationary phase. HPI levels increased in response to ascorbate or H2O2 being added to the medium but HPII levels did not. In minimal medium, any carbon source derived from the tricarboxylic acid cycle caused five- to tenfold higher HPII levels during logarithmic growth but had very little effect on HPI levels. Active electron transport did not affect either HPI or HPII levels.

DNA repair is more important than catalase for Salmonella virulence in mice.

Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites such as hydrogen peroxide (H2O2), which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxify reactive oxygen species, and DNA repair systems which repair damage resulting from oxidative stress. To determine the relative importance of these two potentially protective defense mechanisms against oxidative stress encountered by Salmonella during infection of the host, a Salmonella typhimurium double mutant unable to produce either the HPI or HPII catalase was constructed, and compared with an isogenic recA mutant deficient in DNA repair. The recA mutant was hypersusceptible to H2O2 at low cell densities in vitro, while the catalase mutant was more susceptible to high H2O2 concentrations at high cell densities. The catalase mutant was found to be resistant to macrophages and retained full murine virulence, in contrast to the recA mutant which previously was shown to be macrophage-sensitive and attenuated in mice. These observations suggest that Salmonella is subjected to low concentrations of H2O2 while at relatively low cell density during infection, conditions requiring an intact DNA repair system but not functional catalase activity.

Catalase-peroxidase KatG of Burkholderia pseudomallei at 1.7A resolution.

The catalase-peroxidase encoded by katG of Burkholderia pseudomallei (BpKatG) is 65% identical with KatG of Mycobacterium tuberculosis, the enzyme responsible for the activation of isoniazid as an antibiotic. The structure of a complex of BpKatG with an unidentified ligand, has been solved and refined at 1.7A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are 15.3% and 18.6%, respectively. The crystallized enzyme is a dimer with one modified heme group and one metal ion, likely sodium, per subunit. The modification on the heme group involves the covalent addition of two or three atoms, likely a perhydroxy group, to the secondary carbon atom of the vinyl group on ring I. The added group can form hydrogen bonds with two water molecules that are also in contact with the active-site residues Trp111 and His112, suggesting that the modification may have a catalytic role. The heme modification is in close proximity to an unusual covalent adduct among the side-chains of Trp111, Tyr238 and Met264. In addition, Trp111 appears to be oxidized on C(delta1) of the indole ring. The main channel, providing access of substrate hydrogen peroxide to the heme, contains a region of unassigned electron density consistent with the binding of a pyridine nucleotide-like molecule. An interior cavity, containing the sodium ion and an additional region of unassigned density, is evident adjacent to the adduct and is accessible to the outside through a second funnel-shaped channel. A large cleft in the side of the subunit is evident and may be a potential substrate-binding site with a clear pathway for electron transfer to the active-site heme group through the adduct.

Cloning and physical characterization of katE and katF required for catalase HPII expression in Escherichia coli

Two genes, katE and katF, affecting the synthesis of catalase HPII in Escherichia coli, have been cloned. The multistep cloning protocol involved: screening for the tet gene in a transposon interrupting the genes, selecting DNA adjacent to the transposon, and using it to probe a library of wild-type DNA to select clones from which katE and katF were subcloned into pAT153. The clones were physically characterized and the presence of the genes confirmed by complementation of their respective mutations. The location of the transposon insertions in the two genes was determined by Southern blotting of genomic digests to further confirm the identity of the cloned genes. A 93-kDa protein, the same size as the subunit of HPII, was encoded by the katE plasmid, indicating that katE was the structural gene for HPII. A 44-kDa protein was encoded by the katF plasmid.

Crystal structure of catalase HPII from Escherichia coli

BACKGROUND Catalase is a ubiquitous enzyme present in both the prokaryotic and eukaryotic cells of aerobic organisms. It serves, in part, to protect the cell from the toxic effects of small peroxides. Escherichia coli produces two catalases, HPI and HPII, that are quite distinct from other catalases in physical structure and catalytic properties. HPII, studied in this work, is encoded by the katE gene, and has been characterized as an oligomeric, monofunctional catalase containing one cis-heme d prosthetic group per subunit of 753 residues. RESULTS The crystal structure of catalase HPII from E. coli has been determined to 2.8 A resolution. The asymmetric unit of the crystal contains a whole molecule, which is a tetramer with accurate 222 point group symmetry. In the model built, that includes residues 27-753 and one heme group per monomer, strict non-crystallographic symmetry has been maintained. The crystallographic agreement R-factor is 20.1% for 58,477 reflections in the resolution shell 8.0-2.8 A. CONCLUSIONS Despite differences in size and chemical properties, which were suggestive of a unique catalase, the deduced structure of HPII is related to the structure of catalase from Penicillium vitale, whose sequence is not yet known. In particular, both molecules have an additional C-terminal domain that is absent in the bovine catalase. This extra domain contains a Rossmann fold but no bound nucleotides have been detected, and its physiological role is unknown. In HPII, the heme group is modified to a heme d and inverted with respect to the orientation determined in all previously reported heme catalases. HPII is the largest catalase for which the structure has been determined to almost atomic resolution.

Comparative study of catalase-peroxidases (KatGs).

Catalase-peroxidases or KatGs from seven different organisms, including Archaeoglobus fulgidus,Bacillus stearothermophilus, Burkholderia pseudomallei, Escherichia coli, Mycobacterium tuberculosis, Rhodobacter capsulatus and Synechocystis PCC 6803, have been characterized to provide a comparative picture of their respective properties. Collectively, the enzymes exhibit similar turnover rates with the catalase and peroxidase reactions varying between 4900 and 15,900s(-1) and 8-25s(-1), respectively. The seven enzymes also exhibited similar pH optima for the peroxidase (4.25-5.0) and catalase reactions (5.75), and high sensitivity to azide and cyanide with IC50 values of 0.2-20muM and 50-170muM, respectively. The K(M)s of the enzymes for H2O2 in the catalase reaction were relatively invariant between 3 and 5mM at pH 7.0, but increased to values ranging from 20 to 225mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H2O2 binding to Cpd I. The catalatic k(cat) was 2- to 3-fold higher at pH 5 compared to pH 7, consistent with the uptake of a proton being involved in the reduction of Cpd I. The turnover rates for the INH lyase and isonicotinoyl-NAD synthase reactions, responsible for the activation of isoniazid as an anti-tubercular drug, were also similar across the seven enzymes, but considerably slower, at 0.5 and 0.002s(-1), respectively. Only the NADH oxidase reaction varied more widely between 10(-4) and 10(-2)s(-1) with the fastest rate being exhibited by the enzyme from B. pseudomallei.

Catalase-peroxidases (KatG) Exhibit NADH Oxidase Activity

Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km = 12 μm), but the oxidase reaction is slow (kcat = 0.54 min-1) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG.

Structure of the Heme d of Penicillium vitale and Escherichia coli Catalases

A heme d prosthetic group with the configuration of a cis-hydroxychlorin -spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the α--meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.