Chemical and kinetic reaction mechanisms of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans

Quinohemoprotein amine dehydrogenase (QHNDH) possesses a cysteine tryptophylquinone (CTQ) prosthetic group that catalyzes the oxidative deamination of primary amines. In addition to CTQ, two heme c cofactors are present in QHNDH that mediate the transfer of the substrate-derived electrons from CTQ to an external electron acceptor. Steady-state kinetic assays yielded relatively small k(cat) values (<6 s(-1)), and the rate-limiting step appears to be the interprotein electron transfer from heme in QHNDH to the external electron acceptor. Transient kinetic studies of the CTQ-dependent reduction of heme in QHNDH by amine substrates yielded different rate constants for different substrates (72, 190, and 162 s(-1) for methylamine, butylamine, and benzylamine, respectively). Deuterium kinetic isotope effect (KIE) values of 5.3, 3.9, and 8.5 were observed, respectively, for the reactions of methylamine, butylamine, and benzylamine. These results suggest that the abstraction of a proton from the alpha-methylene group of the substrate, which occurs concomitant with CTQ reduction, is the rate-limiting step in the CTQ-dependent reduction of hemes in QHNDH by these amine substrates. In contrast, the reaction of 2-phenylethylamine with QHNDH does not exhibit a significant KIE ((H)k(3)/(D)k(3) = 1.05) and exhibits a much smaller rate constant of 16 s(-1). This suggests that for 2-phenylethylamine, the rate-limiting step in the single-turnover reaction is either hydrolysis of the imine reaction intermediate from CTQ or product release prior to intraprotein electron transfer. Analysis of the products of the reactions of QHNDH with chiral deuterated 2-phenylethylamines demonstrated that the enzyme abstracts the pro-S proton of the substrate in a highly stereospecific manner. Inspection of the crystal structure of phenylhydrazine-inhibited QHNDH suggests that Asp33(gamma) is the residue that performs the proton abstraction. On the basis of these results, kinetic and chemical reaction mechanisms for QHNDH are proposed and discussed in the context of the crystal structure of the enzyme.     

Sequence-specific and 3'-end selective single-strand DNA binding by the Oxytricha nova telomere end binding protein alpha subunit

Oxytricha nova telomere end binding protein (OnTEBP) specifically recognizes and caps single-strand (T(4)G(4))(2) telomeric DNA at the very 3'-ends of O. nova macronuclear chromosomes. The discovery of proteins homologous to the N-terminal domain of the OnTEBP alpha subunit in Euplotes crassus, Schizosaccharomyces pombe, and Homo sapiens suggests that related proteins are widely distributed in eukaryotes. Previously reported crystal structures of the ssDNA binding domain of the OnTEBP alpha subunit both uncomplexed and complexed with telomeric ssDNA have suggested specific mechanisms for sequence-specific and 3'-end selective recognition of the single-strand telomeric DNA. We now describe comparative binding studies of ssDNA recognition by the N-terminal domain of the OnTEBP alpha subunit. Addition of nucleotides to the 3'-end of the TTTTGGGG telomere repeat decreases the level of alpha binding by up to 7-fold, revealing a modest specificity for a 3'-terminus relative to an internal DNA binding site. Nucleotide substitutions at specific positions within the t(1)t(2)t(3)T(4)G(5)G(6)G(7)G(8) repeat show that base substitutions at some sites do not substantially decrease the binding affinity (<2-fold for lowercase letters), while substitutions at other sites dramatically reduce the binding affinity (>20-fold decrease for the uppercase bold letter). Comparison of the structural and binding data provides unique insights into the ways in which proteins recognize and bind single-stranded DNA.     

Hydrogen exchange-mass spectrometry analysis of beta-amyloid peptide structure

beta-Amyloid peptide (A beta) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration associated with the disease. A beta has proven to be toxic only when aggregated; however, the structure of the aggregated species associated with toxicity is unknown. In the present study, we use hydrogen-deuterium isotope exchange (HX)-electrospray ionization mass spectrometry (MS) along with enzymatic digestion as a tool to examine at near residue level, the changes in A beta structure associated with aggregation to a fibril form. Our results show that the structure of A beta intermediate species formed early in the course of fibrillogenesis is dependent upon solvent conditions. Additionally, the HX-MS data of peptic A beta fragments suggest that the C-terminal segment of the peptide is approximately 35% protected from exchange in fibril-containing samples, relative to monomeric A beta species prepared in DMSO/H(2)O. The N-terminus (residues 1-4) is completely unprotected from exchange, and the fragment containing residues 5-19 is over 50% protected from exchange in the fibril-containing samples. This work contributes to our understanding of A beta structure associated with aggregation and toxicity and further application of this approach may aid in the design of agents that intervene in the A beta aggregation processes associated with neurotoxicity.     

Roles of cytoplasmic osmolytes, water, and crowding in the response of Escherichia coli to osmotic stress: biophysical basis of osmoprotection by glycine betaine

To better understand the biophysical basis of osmoprotection by glycine betaine (GB) and the roles of cytoplasmic osmolytes, water, and macromolecular crowding in the growth of osmotically stressed Escherichia coli, we have determined growth rates and amounts of GB, K(+), trehalose, biopolymers, and water in the cytoplasm of E. coli K-12 grown over a wide range of high external osmolalities (1.02-2.17 Osm) in MOPS-buffered minimal medium (MBM) containing 1 mM betaine (MBM+GB). As osmolality increases, we observe that the amount of cytoplasmic GB increases, the amounts of K(+) (the other major cytoplasmic solute) and of biopolymers remain relatively constant, and the growth rate and the amount of cytoplasmic water decrease strongly, so concentrations of biopolymers and all solutes increase with increasing osmolality. We observe the same correlation between the growth rate and the amount of cytoplasmic water for cells grown in MBM+GB as in MBM, supporting our proposal that the amount of cytoplasmic water is a primary determinant of the growth rate of osmotically stressed cells. We also observe the same correlation between cytoplasmic concentrations of biopolymers and K(+) for cells grown in MBM and MBM+GB, consistent with our hypothesis of compensation between the anticipated large perturbing effects on cytoplasmic protein-DNA interactions of increases in cytoplasmic concentrations of K(+) and biopolymers (crowding) with increasing osmolality. For growth conditions where the amount of cytoplasmic water is relatively large, we find that cytoplasmic osmolality is adequately predicted by assuming that contributions of individual solutes to osmolality are additive and using in vitro osmotic data on osmolytes and a local bulk domain model for cytoplasmic water. At moderate growth osmolalities (up to 1 Osm), we conclude that GB is an efficient osmoprotectant because it is almost as excluded from the biopolymer surface in the cytoplasm as it is from native protein surface in vitro. At very high growth osmolalities where cells contain little cytoplasmic water, predicted cytoplasmic osmolalities greatly exceed observed osmolalities, and the efficiency of GB as an osmolality booster decreases as the amount of cytoplasmic water decreases.     

A microsomal endopeptidase from liver that preferentially degrades stearoyl-CoA desaturase

A protease was purified some 700-fold from rat liver microsomes by a combination of differential detergent solubilization, hydroxyapatite column chromatography, and gel filtration. The protease exhibits substrate selectivity for stearoyl-CoA desaturase (SCD). The purified protease rapidly degraded SCD while other microsomal proteins including cytochrome b(5) and 11beta-hydroxysteroid dehydrogenase were degraded slowly or not at all. The isolated form of the protease has an apparent molecular mass of approximately 90 kDa. Upon incubation, the 90 kDa form of the protease undergoes rapid conversion to a series of smaller proteins. This conversion is associated with a marked increase in proteolytic activity. Diisopropyl phosphofluoridate (DFP) at high concentration partially inhibited the protease activity. The [(3)H]DFP-labeled protease was detected as three protein bands of approximately 66 kDa under nonreducing conditions and a single 25 kDa band under reducing conditions. The purified protease was inhibited by dithiothreitol, suggesting the presence of an essential disulfide bond. These results further define the mechanism by which SCD is rapidly and selectively degraded in isolated liver microsomes.     

Glutamine 132 in the NAD(H)-binding component of proton-translocating transhydrogenase tethers the nucleotides before hydride transfer

Transhydrogenase, found in bacterial membranes and inner mitochondrial membranes of animal cells, couples the redox reaction between NAD(H) and NADP(H) to proton translocation. In this work, the invariant Gln132 in the NAD(H)-binding component (dI) of the Rhodospirillum rubrum transhydrogenase was substituted with Asn (to give dI.Q132N). Mixtures of the mutant protein and the NADP(H)-binding component (dIII) of the enzyme readily produced an asymmetric complex, (dI.Q132N)(2)dIII(1). The X-ray structure of the complex revealed specific changes in the interaction between bound nicotinamide nucleotides and the protein at the hydride transfer site. The first-order rate constant of the redox reaction between nucleotides bound to (dI.Q132N)(2)dIII(1) was <1% of that for the wild-type complex, and the deuterium isotope effect was significantly decreased. The nucleotide binding properties of the dI component in the complex were asymmetrically affected by the Gln-to-Asn mutation. In intact, membrane-bound transhydrogenase, the substitution completely abolished all catalytic activity. The results suggest that Gln132 in the wild-type enzyme behaves as a "tether" or a "tie" in the mutual positioning of the (dihydro)nicotinamide rings of NAD(H) and NADP(H) for hydride transfer during the conformational changes that are coupled to the translocation of protons across the membrane. This ensures that hydride transfer is properly gated and does not take place in the absence of proton translocation.     

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide

The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.     

SLLP1, a unique,intra-acrosomal,non-bacteriolytic,c lysozyme-like protein of human spermatozoa

We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by beta-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P < or = 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, approximately 1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.     

Induction of anti-melanoma CTL response using DC transfected with mutated mRNA encoding full-length Melan-A/MART-1 antigen with an A27L amino acid substitution

Modification of the parental immunodominant Melan-A/MART-1 peptide (MART-1(26-35)) by replacing the alanine with leucine (A27L) enhances its immunogenicity. Because of the reported advantages of RNA over peptides in DC vaccines, we sought to mutate the MART-1 gene to encode a full-length MART-1 antigen with an A27L amino acid substitution. Human DC were transfected with A27L-mutated MART-1 RNA (A27L RNA) or native MART-1 RNA, and then used to stimulate autologous T cells from a series of 8 HLA-A2+ volunteers. After three stimulations, all CTL induced with DC/A27L RNA exhibited more tetramer+ cells, and demonstrated stronger antigen-specific IFNgamma-secreting activity compared to CTL induced with DC/native RNA. A potent MART-1-specific, and predominantly class-I-restricted lysis was detected in most CTL induced with DC/A27L RNA, while native RNA-induced CTL showed minimal and non-specific lysis. HLA-A2+ DC and MART-1 negative/A2+ melanoma cells transfected with the A27L RNA were recognized and killed by MART-1-specific CTL, suggesting that these APC efficiently processed the A27L RNA and presented correct MART-1-specific epitope(s). In summary, introducing an A27L mutation into the MART-1 full-length mRNA sequence enhanced the immunogenicity of the encoded MART-1 Ag. The ease with which such a mutation can be made in RNA presents another potential advantage of using RNA for immunotherapy. Our results support considering this strategy for enhancing the immunogenicity of DC-based RNA vaccines.     

ePAD,an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets

Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.