title: In vitro assembly of catalase.
abstract: 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.
authors.affiliation: From the Microbiology Group, Department of Biology, Lund University, Sölvegatan 35, SE-22362 Lund, Sweden.
authors.forename: Michael
authors.initials: M
authors.lastname: Baureder
authors.affiliation: From the Microbiology Group, Department of Biology, Lund University, Sölvegatan 35, SE-22362 Lund, Sweden.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Barane
authors.affiliation: From the Microbiology Group, Department of Biology, Lund University, Sölvegatan 35, SE-22362 Lund, Sweden lars.hederstedt@biol.lu.se.
authors.forename: Lars
authors.initials: L
authors.lastname: Hederstedt
mesh_terms: D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002374:Catalase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013293:Enterococcus faecalis / Q000737:chemistry* / Q000201:enzymology; D015964:Gene Expression Regulation, Bacterial; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006427:Hemin / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D021122:Protein Subunits; D006418:Heme; D006427:Hemin; D002374:Catalase
keywords: Bacterial Metabolism; Biosynthesis; Catalase; Enterococcus; Enzyme; Heme; Protein Assembly