Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.     

The penicillin-binding site in the exocellular DD-carboxypeptidase-transpeptidase of Actinomadura R39.

Heat denaturation and Pronase degradation of the complex previously formed between benzylpenicillin and the exocellular DD-carboxypeptidase-transpeptidase of Actinomadura R39 yields a heptapeptide H-Leu-Pro-Ala-Ser-Asn-Gly-Val-OH, where the benzylpenicilloyl group is ester-linked to the serine residue. This linkage is very labile and its hydrolysis causes the release of benzylpenicilloate. In contrast, the native benzylpenicilloyl-enzyme complex is very stable (half-life 70 h at 37 degrees C) and its breakdown proceeds via fragmentation of the bound benzylpenicilloyl group [Fuad, Frère, Ghuysen, Duez & Iwatsubo (1976) Biochem. J. 155, 623-629].     

Crystal structure of the 47-kDa lipoprotein of Treponema pallidum reveals a novel penicillin-binding protein

Syphilis is a complex sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. T. pallidum has remained exquisitely sensitive to penicillin, but the mode of action and lethal targets for beta-lactams are still unknown. We previously identified the T. pallidum 47-kDa lipoprotein (Tp47) as a penicillin-binding protein (PBP). Tp47 contains three hypothetical consensus motifs (SVTK, TEN, and KTG) that typically form the active center of other PBPs. Yet, in this study, mutations of key amino acids within these motifs failed to abolish the penicillin binding activity of Tp47. The crystal structure of Tp47 at a resolution of 1.95 A revealed a fold different from any other known PBP; Tp47 is predominantly beta-sheet, in contrast to the alpha/beta-fold common to other PBPs. It comprises four distinct domains: two complex beta-sheet-containing N-terminal domains and two C-terminal domains that adopt immunoglobulin-like folds. The three hypothetical PBP signature motifs do not come together to form a typical PBP active site. Furthermore, Tp47 is unusual in that it displays beta-lactamase activity (k(cat) for penicillin = 271 +/- 6 s(-1)), a feature that hindered attempts to identify the active site in Tp47 by co-crystallization and mass spectrometric techniques. Taken together, Tp47 does not fit the classical structural and mechanistic paradigms for PBPs, and thus Tp47 appears to represent a new class of PBP.     

Crystal structure of the YajQ protein from Haemophilus influenzae reveals a tandem of RNP-like domains

A hypothetical protein encoded by the gene YajQ of Haemophilus influenzae was selected, as part of a structural genomics project, for X-ray crystallographic structure determination and analysis to assist with the functional assignment. The protein is present in most bacteria, but not in archaea or eukaryotes. The amino acid sequence has no homology to that of other proteins.The YajQ protein was cloned, expressed, and the crystal structure determined at 2.1-Å resolution by applying the multiwavelength anomalous dispersion method to a mercury derivative. The polypeptide chain is folded into two domains with identical folding topology. Each domain has a four-stranded antiparallel -sheet flanked on one side by two -helices. This structural motif is a characteristic feature of many RNA-binding proteins. The tetrameric structure observed in the crystal suggests a possibility of binding two stretches of double-stranded nucleic acid.     

Crystal structure of two CD46 domains reveals an extended measles virus-binding surface.

Measles virus is a paramyxovirus which, like other members of the family such as respiratory syncytial virus, is a major cause of morbidity and mortality worldwide. The cell surface receptor for measles virus in humans is CD46, a complement cofactor. We report here the crystal structure at 3.1 A resolution of the measles virus-binding fragment of CD46. The structure reveals the architecture and spatial arrangement of two glycosylated short consensus repeats with a pronounced interdomain bend and some flexibility at the domain interface. Amino acids involved in measles virus binding define a large, glycan-free surface that extends from the top of the first to the bottom of the second repeat. The extended virus-binding surface of CD46 differs strikingly from those reported for the human virus receptor proteins CD4 and intercellular cell adhesion molecule-1 (ICAM-1), suggesting that the CD46 structure utilizes a novel mode of virus recognition. A highly hydrophobic and protruding loop at the base of the first repeat bears a critical virus-binding residue, thereby defining an important recognition epitope. Molecules that mimic the conformation of this loop potentially could be effective anti-viral agents by preventing binding of measles virus to CD46.     

Interaction of clavulanate with the beta-lactamases of Streptomyces albus G and Actinomadura R39.

The reactions of beta-lactamases of Actinomadura R39 and Streptomyces albus G with clavulanate proceed along branched pathways. Both enzymes perform the hydrolysis of this beta-lactam with rather high efficiencies (kcat. = 18s-1 and 52s-1 respectively). If large clavulanate/enzyme ratios are used, complete inactivation of the enzymes is observed. At lower ratios, inactivation is only partial. Irreversible inactivation occurs after 400 and 20000 turnovers for the A. R39 and S. albus G enzymes respectively. With the A. R39 beta-lactamase, a transiently inhibited complex is also formed that remains undetectable with the S. albus G beta-lactamase. Kinetic models are presented and studied for the interaction between clavulanate and both enzymes. A tentative general reaction scheme is also discussed.     

Interaction of beta-iodopenicillanate with the beta-lactamases of Streptomyces albus G and Actinomadura R39.

The beta-lactamases of Streptomyces albus G and Actinomadura R39 are inactivated by beta-iodopenicillanate. However, in contrast with the beta-lactamase I from Bacillus cereus, they also efficiently catalyse the hydrolysis of the inactivator; with the S. albus G enzyme, kcat. is larger than 25s-1 and the number of turnovers before inactivation is 515. With the A. R39 enzyme, kcat. is larger than 50s-1 and the number of turnovers before inactivation is 80. After hydrolysis of the beta-lactam amide bond, the product rearranges into 2.3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which exhibits an absorption maximum at 305 nm.     

Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin

The interaction between ubiquitinated proteins and intracellular proteins harboring ubiquitin binding domains (UBDs) is critical to a multitude of cellular processes. Here, we report that Rabex-5, a guanine nucleotide exchange factor for Rab5, binds to Ub through two independent UBDs. These UBDs determine a number of properties of Rabex-5, including its coupled monoubiquitination and interaction in vivo with ubiquitinated EGFRs. Structural and biochemical characterization of the UBDs of Rabex-5 revealed that one of them (MIU, motif interacting with ubiquitin) binds to Ub with modes superimposable to those of the UIM (ubiquitin-interacting motif):Ub interaction, although in the opposite orientation. The other UBD, RUZ (Rabex-5 ubiquitin binding zinc finger) binds to a surface of Ub centered on Asp58(Ub) and distinct from the "canonical" Ile44(Ub)-based surface. The two binding surfaces allow Ub to interact simultaneously with different UBDs, thus opening new perspectives in Ub-mediated signaling.     

Crystal structure of the BARD1 BRCT domains

The interaction of the breast tumor suppressor BRCA1 with the protein BARD1 results in the formation of a heterodimeric complex that has ubiquitin ligase activity and plays central roles in cell cycle checkpoint control and DNA repair. Both BRCA1 and BARD1 possess a pair of tandem BRCT domains that interact in a phosphorylation-dependent manner with target proteins. We determined the crystal structure of the human BARD1 BRCT repeats (residues 568-777) at 1.9 A resolution. The composition and structure of the BARD1 phosphoserine-binding pocket P1 are strikingly similar to those of the BRCA1 and MDC1 BRCT domains, suggesting a similar mode of interaction with the phosphate group of the ligand. By contrast, the BARD1 BRCT selectivity pocket P2 exhibits distinct structural features, including two prominent histidine residues, His685 and His686, which may be important for ligand binding. The protonation state of these histidines has a marked effect on the calculated electrostatic potential in the vicinity of P2, raising the possibility that ligand recognition may be regulated by changes in pH. Importantly, the BARD1 BRCT structure provides insights into the mechanisms by which the cancer-associated missense mutations C645R, V695L, and S761N may adversely affect the structure and function of BARD1.     

The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis

Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.     

Primary structure of the Streptomyces R61 extracellular DD-peptidase. 2. Amino acid sequence data

In order to confirm the Streptomyces codon usage, the Streptomyces R61 DD-peptidase was fragmented by cyanogen bromide cleavage of the carboxymethylated protein, trypsin digestion of the carboxymethylated protein and trypsin digestion of the protein treated with beta-iodopenicillinate and endoxo-delta 4-tetrahydrophthalic acid. The isolated peptides, which altogether represented more than 50% of the polypeptide chain, were sequenced. The data thus obtained were in excellent agreement with the primary structure of the protein as deduced from the nucleotide sequence of the cloned gene. Though a weak acylating agent, beta-iodopenicillanate reacted selectively with the active site of the DD-peptidase and formed an adduct which mas much more stable than that formed with benzylpenicillin, thus facilitating the isolation and characterization of the active-site peptide.     

Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site

We report crystal structures of the human enzyme phosphoenolpyruvate carboxykinase (PEPCK) with and without bound substrates. These structures are the first to be determined for a GTP-dependent PEPCK, and provide the first view of a novel GTP-binding site unique to the GTP-dependent PEPCK family. Three phenylalanine residues form the walls of the guanine-binding pocket on the enzyme's surface and, most surprisingly, one of the phenylalanine side-chains contributes to the enzyme's specificity for GTP. PEPCK catalyzes the rate-limiting step in the metabolic pathway that produces glucose from lactate and other precursors derived from the citric acid cycle. Because the gluconeogenic pathway contributes to the fasting hyperglycemia of type II diabetes, inhibitors of PEPCK may be useful in the treatment of diabetes.     

Crystal structure of hemopexin reveals a novel high-affinity heme site formed between two beta-propeller domains.

The ubiquitous use of heme in animals poses severe biological and chemical challenges. Free heme is toxic to cells and is a potential source of iron for pathogens. For protection, especially in conditions of trauma, inflammation and hemolysis, and to maintain iron homeostasis, a high-affinity binding protein, hemopexin, is required. Hemopexin binds heme with the highest affinity of any known protein, but releases it into cells via specific receptors. The crystal structure of the heme-hemopexin complex reveals a novel heme binding site, formed between two similar four-bladed beta-propeller domains and bounded by the interdomain linker. The ligand is bound to two histidine residues in a pocket dominated by aromatic and basic groups. Further stabilization is achieved by the association of the two beta-propeller domains, which form an extensive polar interface that includes a cushion of ordered water molecules. We propose mechanisms by which these structural features provide the dual function of heme binding and release.     

Crystal structure of the kinase and UBA domains of SNRK reveals a distinct UBA binding mode in the AMPK family

Sucrose non-fermenting (Snf1)-related kinase (SNRK) is a novel member of the AMP-activated protein kinase (AMPK) family and is involved in many metabolic processes. Here we report the crystal structure of an N-terminal SNRK fragment containing kinase and adjacent ubiquitin-associated (UBA) domains. This structure shows that the UBA domain binds between the N- and C-lobes of the kinase domain. The mode of UBA binding in SNRK largely resembles that in AMPK and brain specific kinase (BRSK), however, unique interactions play vital roles in stabilizing the KD-UBA interface of SNRK. We further propose a potential role of the UBA domain in the regulation of SNRK kinase activity. This study provides new insights into the structural diversities of the AMPK kinase family.     

Nucleotide sequence of the gene encoding the active-site serine β-lactamase from Actinomadura R39

The gene encoding the extracellular, active-site serine beta-lactamase of Actinomadura R39, previously cloned into Streptomyces lividans, has the information for the synthesis of a 304 amino acid protein, the amino terminal region of which has the characteristic features of a signal peptide. The Actinomadura R39 beta-lactamase is another member of the class A beta-lactamases. In particular, it shows high homology with the beta-lactamase of Bacillus licheniformis.     

Brave new proteins: what evolution reveals about protein structure.

Synthetic proteins provide important information about the principles of protein structure. They illuminate the processes of natural protein evaluation, but are not limited by these processes. Here, we review studies of several mutant proteins and discuss the general principles that can be derived from them.