Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process.

Lactic acid bacteria (LAB) are of profound importance in food production and infection medicine. LAB do not rely on heme (protoheme IX) for growth and are unable to synthesize this cofactor but are generally able to assemble a small repertoire of heme-containing proteins if heme is provided from an exogenous source. These features are in contrast to other bacteria, which synthesize their heme or depend on heme for growth. We here present the cellular function of heme proteins so far identified in LAB and discuss their biogenesis as well as applications of the extraordinary heme physiology of LAB.

Monoamine oxidase-B (Mao-B) catalysing the breakdown of the neurotransmitter dopamine, is known to be involved in the pathophysiology of Parkinson's (PD) and Alzheimer's disease (AD). Increased brain Mao-B activity is associated with AD. This alteration can also be seen in platelets, albeit the cause has hitherto remained elusive. To gain a deeper understanding of the etiology of AD, the platelet proteome was characterised, (2D DIGE pH6-9, including Mao-B) from 150 individuals: 34 AD, 13 vascular dementia, 15 non-demented PD patients, 49 matched controls, 18 oldest old and 21 young individuals. One significant change was noted after applying false discovery rate with the upregulation of the Mao-B expression (30% adjusted P value < 0.001; effect size 1.31) in AD compared to age- and sex-matched controls. In contrast, Mao-B levels were unchanged in PD to matched controls. Western blot and mRNA analyses verified these findings. Moreover, Mao-B concentration correlated with age in the cognitive healthy individuals (r = 0.53; P < 0.001) and PD patients but not in those suffering from AD (r = − 0.03; P = 0.874). Mao-B activity correlated with the increased Mao-B protein expression in AD (r = 0.81; P = 0.016). We suggest that Mao-B platelet protein level may serve as a biomarker for age-related dementia, especially AD.

Enterococcus faecalis exhibits high resistance to oxidative stress. Several enzymes are responsible for this trait. The role of alkyl hydroperoxide reductase (Ahp), thiol peroxidase (Tpx), and NADH peroxidase (Npr) in oxidative stress defense was recently characterized. Enterococcus faecalis, in contrast to many other streptococci, contains a catalase (KatA), but this enzyme can only be formed when the bacterium is supplied with heme. We have used this heme dependency of catalase activity and mutants deficient in KatA and Npr to investigate the role of the catalase in resistance against exogenous and endogenous hydrogen peroxide stress. The results demonstrate that in the presence of environmental heme catalase contributes to the protection against toxic effects of hydrogen peroxide.

Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly.

Heme proteins form a large and diverse group of proteins which are involved in a variety of biological functions. The heme prosthetic group enables them to carry out redox reactions, transport electrons, bind gaseous molecules, and function as sensors. Despite their importance only little is generally known about heme protein biogenesis. The Gram-positive bacterium Enterococcus faecalis is found in the gastrointestinal tract of mammals and is an opportunistic pathogen. E. faecalis cannot synthesize heme and does not require heme for growth. When supplied with heme, this bacterium produces two heme proteins; one catalase and one cytochrome bd. These properties of E. faecalis have been exploited in this work to study the physiological role and biogenesis of catalase. Catalase was found to contribute to resistance against exogenous and endogenous hydrogen peroxide stress. It is shown that the gene for catalase, katA, is expressed independently of heme in the growth medium. KatA protein was found in cells growing in heme-free medium but was degraded in stationary growth phase unless heme was supplied. These and other findings were used to devise a procedure for the purification of apo-catalase polypeptide. It is demonstrated in vitro with isolated apo-catalase and heme that catalase can be de novo assembled. The obtained catalase contained stoichiometric amounts of heme but did not show full enzyme activity. These and other results suggested that the in vitro assembled catalase is stalled at an intermediate state and that one or more soluble cell factors are needed to complete assembly or activation of the enzyme. To find novel factors important for catalase assembly, two constructed libraries of transposon-insertion mutants were screened for catalase deficient mutants. In this way ten independent katA mutations were isolated but no factors (genes) with a dedicated essential role for catalase biogenesis or heme trafficking were revealed. However, the screen indicated nine genes, distributed over five different chromosomal loci, which apparently indirectly affect expression of catalase in E. faecalis.

Cytochrome b₅₅₈ of the gram-positive bacterium Bacillussubtilis is the membrane anchor subunit of the succinate:quinone oxidoreductase of the citric acid cycle. The cytochrome consists of the SdhC polypeptide (202 residues) and two protoheme IX groups that function in transmembrane electron transfer to menaquinone. The general structure of the cytochrome is known from extensive experimental studies and by comparison to Wolinellasuccinogenes fumarate reductase for which the X-ray crystal structure has been determined. Solution state NMR can potentially be used to identify the quinone binding site(s) and study, e.g. redox-linked, dynamics of cytochrome b₅₅₈. In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome b₅₅₈ produced in Escherichia coli. We have also evaluated several detergents suitable for NMR for their effectiveness in maintaining the cytochrome solubilized and intact for days at room temperature.