Protein acetylation is a widespread modification that is mediated by site-selective acetyltransferases. KATs (lysine N ⑀ -acetyltransferases), modify the side chain of specific lysines on histones and other proteins, a central process in regulating gene expression. N ␣ -terminal acetylation occurs on the ribosome where the ␣ amino group of nascent polypeptides is acetylated by NATs (N-terminal acetyltransferase). In yeast, three different NAT complexes were identified NatA, NatB, and NatC. NatA is composed of two main subunits, the catalytic subunit Naa10p (Ard1p) and Naa15p (Nat1p). Naa50p (Nat5) is physically associated with NatA. In man, hNaa50p was shown to have acetyltransferase activity and to be important for chromosome segregation. In this study, we used purified recombinant hNaa50p and multiple oligopeptide substrates to identify and characterize an N ␣ -acetyltransferase activity of hNaa50p. As the preferred substrate this activity acetylates oligopeptides with N termini Met-Leu-Xxx-Pro. Furthermore, hNaa50p autoacetylates lysines 34, 37, and 140 in vitro, modulating hNaa50p substrate specificity. In addition, histone 4 was detected as a hNaa50p KAT substrate in vitro. Our findings thus provide the first experimental evidence of an enzyme having both KAT and NAT activities.

High-resolution two-dimensional gel electrophoresis and mass spectrometry has been used to identify the outer membrane (OM) subproteome of the Gram-negative bacterium Methylococcus capsulatus (Bath). Twenty-eight unique polypeptide sequences were identified from protein samples enriched in OMs. Only six of these polypeptides had previously been identified. The predictions from novel bioinformatic methods predicting beta-barrel outer membrane proteins (OMPs) and OM lipoproteins were compared to proteins identified experimentally. BOMP ( http://www.bioinfo.no/tools/bomp ) predicted 43 beta-barrel OMPs (1.45%) from the 2,959 annotated open reading frames. This was a lower percentage than predicted from other Gram-negative proteomes (1.8-3%). More than half of the predicted BOMPs in M. capsulatus were annotated as (conserved) hypothetical proteins with significant similarity to very few sequences in Swiss-Prot or TrEMBL. The experimental data and the computer predictions indicated that the protein composition of the M. capsulatus OM subproteome was different from that of other Gram-negative bacteria studied in a similar manner. A new program, Lipo, was developed that can analyse entire predicted proteomes and give a list of recognised lipoproteins categorised according to their lipo-box similarity to known Gram-negative lipoproteins ( http://www.bioinfo.no/tools/lipo ). This report is the first using a proteomics and bioinformatics approach to identify the OM subproteome of an obligate methanotroph.

No abstract

SummaryIdentification of surface proteins is essential to understand bacterial communication with its environment. Analysis of the surface-associated proteins of Methylococcus capsulatus (Bath) revealed a highly dynamic structure responding closely to the availability of copper in the medium in the range from 0 to 10 mM. Several c-type cytochromes, including three novel multihaem proteins, are present at the cellular surface, a feature that is otherwise a peculiarity of dissimilatory metal-reducing bacteria. At low copper concentrations, the cytochrome c 553o and the cytochrome c553o family protein, encoded by the MCA0421 and MCA0423 genes, respectively, are major constituents of the surfaceome and show a fine-tuned copper-dependent regulation of expression. Two novel members of the cytochrome c553o family were identified: MCA0338 was abundant between 5 and 10 mM copper, while MCA2259 was detected only in the surface fraction obtained from 0 mM copper cultures. The presence at the bacterial surface of several c-type cytochromes, generally involved in energy transduction, indicates strongly that redox processes take place at the bacterial surface. Due to the unique role of copper in the biology of M. capsulatus (Bath), it appears that c-type cytochromes have essential functions in copper homeostasis allowing the cells to adapt to varying copper exposure.

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Haemerythrin proteins comprise a family of O 2 -carrrying proteins mainly found in a few phyla of marine invertebrates. Members of this family differ from haemoglobin and haemocyanin in that they contain a nonheme diiron site that reversibly binds one molecule of O 2 . This oxygen-binding binuclear iron complex is a characteristic feature of haemerythrins. The two iron ions are bound to the protein via seven conserved amino acid residues; five histidines, one glutamate and one aspartate [1]. All known haemerythrins also share a four-helix bundle fold which surrounds the diiron site. Furthermore, the haemerythrins are divided into two subfamilies; the haemerythrins (Hr) and myohaemerythrins (MHr). Hrs are found in coelomic cells and typically exist as homopolymers composed of subunits of 113-117 amino acid residues. MHrs are For a long time, the haemerythrin family of proteins was considered to be restricted to only a few phyla of marine invertebrates. When analysing differential protein expression in the methane-oxidizing bacterium, Methylococcus capsulatus (Bath), grown at a high and low copper-to-biomass ratio, respectively, we identified a putative prokaryotic haemerythrin expressed in high-copper cultures. Haemerythrins are recognized by a conserved sequence motif that provides five histidines and two carboxylate ligands which coordinate two iron atoms. The diiron site is located in a hydrophobic pocket and is capable of binding O 2 . We cloned the M. capsulatus haemerythrin gene and expressed it in Escherichia coli as a fusion protein with NusA. The haemerythrin protein was purified to homogeneity cleaved from its fusion partner. Recombinant M. capsulatus haemerythrin (McHr) was found to fold into a stable protein. Sequence similarity analysis identified all the candidate residues involved in the binding of diiron (His22, His58, Glu62, His77, His81, His117, Asp122) and the amino acids forming the hydrophobic pocket in which O 2 may bind (Ile25, Phe59, Trp113, Leu114, Ile118). We were also able to model a three-dimensional structure of McHr maintaining the correct positioning of these residues. Furthermore, UV ⁄ vis spectrophotometric analysis demonstrated the presence of conjugated diiron atoms in McHr. A comprehensive genomic database search revealed 21 different prokaryotes containing the haemerythrin signature (PROSITE 00550), indicating that these putative haemerythrins may be a conserved prokaryotic subfamily.Abbreviations 2DE, two-dimensional gel electrophoresis; Hr, haemerythrin; ICP-MS, inductively coupled plasma atomic emission-mass spectrometry; IPG, immobilized pH gradient; IPTG, isopropyl thio-b-D-galactoside; McHr, Methylococcus capsulatus haemerythrin; MHr, myohaemerythrin; MMO, methane monooxygenase; NMS, nitrate mineral salt; pMMO, particulate methane monooxygenase; sMMO, soluble methane monooxygenase.

Expression of surface-associated and secreted protein MopE of the methanotrophic bacterium Methylococcus capsulatus (Bath) in response to the concentration of copper ions in the growth medium was investigated. The level of protein associated with the cells and secreted to the medium changed when the copper concentration in the medium varied and was highest in cells exposed to copper stress

Copper plays a very significant role in the physiology of the methanotrophic bacterium Methylococcus capsulatus (Bath). The availability of this metal ion regulates expression of the two forms of the methane-oxidizing enzyme methane monooxygenase (MMO) the bacterium possesses and formation of an extensive intracytoplasmic membrane network [1][2][3][4]. When copper is scarce, at a low copper-to-biomass ratio, a soluble cytoplasmic MMO (sMMO) is responsible for the oxidation of methane. At high copper-to-biomass ratios the particulate MMO (pMMO) is expressed and there is no detectable sMMO expression. Furthermore, copper also influences the expression of at least two of the four M. capsulatus formaldehyde dehydrogenases [5][6][7].