Structural Insights into the Substrate Specificity of the Rhodopseudomonas palustris Protein Acetyltransferase RpPat: IDENTIFICATION OF A LOOP CRITICAL FOR RECOGNITION BY RpPat*?

Lysine acetylation is a post-translational modification that is important for the regulation of metabolism in both prokaryotes and eukaryotes. In bacteria, the best studied protein acetyltransferase is Pat. In the purple photosynthetic bacterium Rhodopseudomonas palustris, at least 10 AMP-forming acyl-CoA synthetase enzymes are acetylated by the Pat homologue RpPat. All bona fide RpPat substrates contain the conserved motif PX₄GK. Here, we show that the presence of such a motif is necessary but not sufficient for recognition by RpPat. RpPat failed to acetylate the methylmalonyl-CoA synthetase of this bacterium (hereafter RpMatB) in vivo and in vitro, despite the homology of RpMatB to known RpPat substrates. We used RpMatB to identify structural determinants that are recognized by RpPat. To do this, we constructed a series of RpMatB chimeras that became substrates of RpPat. In such chimeras, a short region (11–25 residues) of RpMatB located >20 residues N-terminal to the acetylation site was replaced with the corresponding sequences from other AMP-forming acyl-CoA synthetases that were known RpPat substrates. Strikingly, the enzymatic activity of RpMatB chimeras was regulated by acetylation both in vitro and in vivo. Crystal structures of two of these chimeras showed that the major difference between them and wild-type RpMatB was within a loop region ∼23 Å from the acetylation site. On the basis of these results, we suggest that RpPat likely interacts with a relatively large surface of its substrates, in addition to the PX₄GK motif, and that RpPat probably has relatively narrow substrate specificity.     

Electron microscopic immunogold localization of salivary mucins MG1 and MG2 in human submandibular and sublingual glands.

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.     

Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

We describe the construction and use of two sets of vectors for the over-expression and purification of protein from Escherichia coli. The set of pTEV plasmids (pTEV3, 4, 5) directs the synthesis of a recombinant protein with a N-terminal hexahistidine (His(6)) tag that is removable by the tobacco etch virus (TEV) protease. The set of pKLD plasmids (pKLD66, 116) directs the synthesis of a recombinant protein that contains a N-terminal His(6) and maltose-binding protein tag in tandem, which can also be removed with TEV protease. The usefulness of these plasmids is illustrated by the rapid, high-yield purification of the 2-methylcitrate dehydratase (PrpD) protein of Salmonella enterica, and the 2-methylaconitate isomerase (PrpF) protein of Shewanella oneidensis, two enzymes involved in the catabolism of propionate to pyruvate via the 2-methylcitric acid cycle.     

Three-dimensional structure of ATP:corrinoid adenosyltransferase from Salmonella typhimurium in its free state, complexed with MgATP, or complexed with hydroxycobalamin and MgATP

In Salmonella typhimurium, formation of the cobalt-carbon bond in the biosynthetic pathway for adenosylcobalamin is catalyzed by the product of the cobA gene which encodes a protein of 196 amino acid residues. This enzyme is an ATP:co(I)rrinoid adenosyltransferase which transfers an adenosyl moiety from MgATP to a broad range of co(I)rrinoid substrates that are believed to include cobinamide, its precursor cobyric acid and probably others as yet unidentified, and hydroxocobalamin. Three X-ray structures of CobA are reported here: its substrate-free form, a complex of CobA with MgATP, and a ternary complex of CobA with MgATP and hydroxycobalamin to 2.1, 1.8, and 2.1 A resolution, respectively. These structures show that the enzyme is a homodimer. In the apo structure, the polypeptide chain extends from Arg(28) to Lys(181) and consists of an alpha/beta structure built from a six-stranded parallel beta-sheet with strand order 324516. The topology of this fold is very similar to that seen in RecA protein, helicase domain, F(1)ATPase, and adenosylcobinamide kinase/adenosylcobinamide guanylyltransferase where a P-loop is located at the end of the first strand. Strikingly, the nucleotide in the MgATP.CobA complex binds to the P-loop of CobA in the opposite orientation compared to all the other nucleotide hydrolases. That is, the gamma-phosphate binds at the location normally occupied by the alpha-phosphate. The unusual orientation of the nucleotide arises because this enzyme transfers an adenosyl group rather than the gamma-phosphate. In the ternary complex, the binding site for hydroxycobalamin is located in a shallow bowl-shaped depression at the C-terminal end of the beta-sheet of one subunit; however, the active site is capped by the N-terminal helix from the symmetry-related subunit that now extends from Gln(7) to Ala(24). The lower ligand of cobalamin is well-ordered and interacts mostly with the N-terminal helix of the symmetry-related subunit. Interestingly, there are few interactions between the protein and the polar side chains of the corrin ring which accounts for the broad specificity of this enzyme. The corrin ring is oriented such that the cobalt atom is located approximately 6.1 A from C5' of the ribose and is beyond the range of nucleophilic attack. This suggests that a conformational change occurs in the ternary complex when Co(III) is reduced to Co(I).     

Chemical force microscopy with active enzymes

The adhesion forces have been measured between an atomic force microscope tip derivatized with an active enzyme, shikimate kinase, and an ATP mimic immobilized on a gold surface. Experiments with competitive binding of other ligands in solution show that the observed adhesion forces arise predominantly from specific interactions between the immobilized enzyme and surface-bound adenine derivative. These experiments represent a step in the development of a screening methodology based upon chemical force microscopy.     

Hybrid scanning ion conductance and scanning near-field optical microscopy for the study of living cells

We have developed a hybrid scanning ion conductance and scanning near-field optical microscope for the study of living cells. The technique allows quantitative, high-resolution characterization of the cell surface and the simultaneous recording of topographic and optical images. A particular feature of the method is a reliable mechanism to control the distance between the probe and the sample in physiological buffer. We demonstrate this new method by recording near-field images of living cells (cardiac myocytes).     

X-ray structures of the Dictyostelium discoideum myosin motor domain with six non-nucleotide analogs

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with dinitrophenylaminoethyl-, dinitrophenylaminopropyl-, o-nitrophenylaminoethyl-, m-nitrophenylaminoethyl-, p-nitrophenylaminoethyl-, and o-nitrophenyl-N-methyl-aminoethyl-diphosphate.beryllium fluoride have been determined to better than 2.3-A resolution. The structure of the protein and nucleotide binding pocket in these complexes is very similar to that of S1dC.ADP.BeF(x) (Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960-8972). The position of the triphosphate-like moiety is essentially identical in all complexes. Furthermore, the alkyl-amino group plays the same role as the ribose by linking the triphosphate to the adenine binding pocket; however, none of the phenyl groups lie in the same position as adenine in S1dC.MgADP.BeF(x), even though several of these nucleotide analogs are functionally equivalent to ATP. Rather the former location of adenine is occupied by water in the nanolog complexes, and the phenyl groups are organized in a manner that attempts to optimize their hydrogen bonding interactions with this constellation of solvent molecules. A comparison of the kinetic and structural properties of the nanologs relative to ATP suggests that the ability of a substrate to sustain tension and to generate movement correlates with a well defined interaction with the active site water structure observed in S1dC.MgADP.BeF(x).     

Evolution of enzymatic activities in the enolase superfamily: crystallographic and mutagenesis studies of the reaction catalyzed by D-glucarate dehydratase from Escherichia coli

D-Glucarate dehydratase (GlucD) from Escherichia coli catalyzes the dehydration of both D-glucarate and L-idarate as well as their interconversion via epimerization. GlucD is a member of the mandelate racemase (MR) subgroup of the enolase superfamily, the members of which catalyze reactions that are initiated by abstraction of the alpha-proton of a carboxylate anion substrate. Alignment of the sequence of GlucD with that of MR reveals a conserved Lys-X-Lys motif and a His-Asp dyad homologous to the S- and R-specific bases in the active site of MR. Crystals of GlucD have been obtained into which the substrate D-glucarate and two competitive inhibitors, 4-deoxy-D-glucarate and xylarohydroxamate, could be diffused; D-glucarate is converted to the dehydration product, 5-keto-4-deoxy-D-glucarate (KDG). The structures of these complexes have been determined and reveal the identities of the ligands for the required Mg(2+) (Asp(235), Glu(266), and Asn(289)) as well as confirm the expected presence of Lys(207) and His(339), the catalytic bases that are properly positioned to abstract the proton from C5 of L-idarate and D-glucarate, respectively. Surprisingly, the C6 carboxylate group of KDG is a bidentate ligand to the Mg(2+), with the resulting geometry of the bound KDG suggesting that stereochemical roles of Lys(207) and His(339) are reversed from the predictions made on the basis of the established structure-function relationships for the MR-catalyzed reaction. The catalytic roles of these residues have been examined by characterization of mutant enzymes, although we were unable to use these to demonstrate the catalytic independence of Lys(207) and His(339) as was possible for the homologous Lys(166) and His(297) in the MR-catalyzed reaction.