Biochemical Characterization of Signal Peptidase I from Gram-Positive Streptococcus pneumoniae

ABSTRACT Bacterial signal peptidase I is responsible for proteolytic processing of the precursors of secreted proteins. The enzymes from gram-negative and -positive bacteria are different in structure and specificity. In this study, we have cloned, expressed, and purified the signal peptidase I of gram-positive Streptococcus pneumoniae. The precursor of streptokinase, an extracellular protein produced in pathogenic streptococci, was identified as a substrate of S. pneumoniae signal peptidase I. Phospholipids were found to stimulate the enzymatic activity. Mutagenetic analysis demonstrated that residues serine 38 and lysine 76 of S. pneumoniae signal peptidase I are critical for enzyme activity and involved in the active site to form a serine-lysine catalytic dyad, which is similar to LexA-like proteases andEscherichia coli signal peptidase I. Similar to LexA-like proteases, S. pneumoniae signal peptidase I catalyzes an intermolecular self-cleavage in vitro, and an internal cleavage site has been identified between glycine 36 and histidine 37. Sequence analysis revealed that the signal peptidase I and LexA-like proteases show sequence homology around the active sites and some common properties around the self-cleavage sites. All these data suggest that signal peptidase I and LexA-like proteases are closely related and belong to a novel class of serine proteases.

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In vitro and in vivo self-cleavage of Streptococcus pneumoniae signal peptidase I

European Journal of Biochemistry ◽

10.1046/j.1432-1033.2002.03083.x ◽

2002 ◽

Vol 269 (16) ◽

pp. 3969-3977 ◽

Cited By ~ 6

Author(s):

Feng Zheng ◽

Eddie L. Angleton ◽

Jin Lu ◽

Sheng-Bin Peng

Keyword(s):

Streptococcus Pneumoniae ◽

Signal Peptidase ◽

Signal Peptidase I

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Biochemical Characterization of Streptococcus pneumoniae Penicillin-Binding Protein 2b and Its Implication in β-Lactam Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.5.1848-1855.2004 ◽

2004 ◽

Vol 48 (5) ◽

pp. 1848-1855 ◽

Cited By ~ 26

Author(s):

Estelle Pagliero ◽

Laurent Chesnel ◽

Julie Hopkins ◽

Jacques Croizé ◽

Otto Dideberg ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Biochemical Characterization ◽

Protein A ◽

Strain Analysis ◽

Penicillin G ◽

Penicillin Binding Protein ◽

Class B ◽

Selection Of

ABSTRACT Extensive use of β-lactam antibiotics has led to the selection of pathogenic streptococci resistant to β-lactams due to modifications of the penicillin-binding proteins (PBPs). PBP2b from Streptococcus pneumoniae is a monofunctional (class B) high-molecular-weight PBP catalyzing the transpeptidation between adjacent stem peptides of peptidoglycan. The transpeptidase domain of PBP2b isolated from seven clinical resistant (CR) strains contains 7 to 44 amino acid changes over the sequence of PBP2b from the R6 β-lactam-sensitive strain. We show that the extracellular soluble domains of recombinant PBP2b proteins (PBP2b*) originating from these CR strains have an in vitro affinity for penicillin G that is reduced by up to 99% from that of the R6 strain. The Thr446Ala mutation is always observed in CR strains and is close to the key conserved motif (S443SN). The Thr446Ala mutation in R6 PBP2b* displays a 60% reduction in penicillin G affinity in vitro compared to that for the wild-type protein. A recombinant R6 strain expressing the R6 PBP2b Thr446Ala mutation is twofold less sensitive to piperacillin than the parental S. pneumoniae strain. Analysis of the Thr446Ala mutation in the context of the PBP2b CR sequences revealed that its influence depends upon the presence of other unidentified mutations.

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Spectrum and characterization of bi-allelic variants in MMAB causing cblB-type methylmalonic aciduria

Human Genetics ◽

10.1007/s00439-021-02398-6 ◽

2021 ◽

Author(s):

Patrick Forny ◽

Tanja Plessl ◽

Caroline Frei ◽

Celine Bürer ◽

D. Sean Froese ◽

...

Keyword(s):

Active Sites ◽

Biochemical Characterization ◽

Isothermal Calorimetry ◽

Disease Onset ◽

Autosomal Recessive Disorder ◽

Methylmalonic Aciduria ◽

Pathogenic Variants ◽

Novel Variants

AbstractPathogenic variants in MMAB cause cblB-type methylmalonic aciduria, an autosomal-recessive disorder of propionate metabolism. MMAB encodes ATP:cobalamin adenosyltransferase, using ATP and cob(I)alamin to create 5’-deoxyadenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase (MMUT). We identified bi-allelic disease-causing variants in MMAB in 97 individuals with cblB-type methylmalonic aciduria, including 33 different and 16 novel variants. Missense changes accounted for the most frequent pathogenic alleles (p.(Arg186Trp), N = 57; p.(Arg191Trp), N = 19); while c.700C > T (p.(Arg234*)) was the most frequently identified truncating variant (N = 14). In fibroblasts from 76 affected individuals, the ratio of propionate incorporation in the presence and absence of hydroxocobalamin (PI ratio) was associated to clinical cobalamin responsiveness and later disease onset. We found p.(Arg234*) to be associated with cobalamin responsiveness in vitro, and clinically with later onset; p.(Arg186Trp) and p.(Arg191Trp) showed no clear cobalamin responsiveness and early onset. Mapping these and novel variants onto the MMAB structure revealed their potential to affect ATP and AdoCbl binding. Follow-up biochemical characterization of recombinant MMAB identified its three active sites to be equivalent for ATP binding, determined by fluorescence spectroscopy (Kd = 21 µM) and isothermal calorimetry (Kd = 14 µM), but function as two non-equivalent AdoCbl binding sites (Kd1 = 0.55 μM; Kd2 = 8.4 μM). Ejection of AdoCbl was activated by ATP (Ka = 24 µM), which was sensitized by the presence of MMUT (Ka = 13 µM). This study expands the landscape of pathogenic MMAB variants, provides association of in vitro and clinical responsiveness, and facilitates insight into MMAB function, enabling better disease understanding.

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Residues flanking the COOH-terminal C-region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase and the extent of coupling of its co-translational translocation and proteolytic processing in vitro.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)45810-3 ◽

1990 ◽

Vol 265 (35) ◽

pp. 21797-21803

Author(s):

S F Nothwehr ◽

S D Hoeltzli ◽

K L Allen ◽

M O Lively ◽

J I Gordon

Keyword(s):

Signal Peptide ◽

Proteolytic Processing ◽

Signal Peptidase

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Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

Veterinary Pathology ◽

10.1177/030098588502200413 ◽

1985 ◽

Vol 22 (4) ◽

pp. 375-386 ◽

Cited By ~ 6

Author(s):

H. C. Wimberly ◽

D. O. Slauson ◽

N. R. Neilsen

Keyword(s):

Functional Activity ◽

Biochemical Characterization ◽

Platelet Activating Factor ◽

Biochemical Properties ◽

Naturally Occurring ◽

Rabbit Platelets ◽

Rapid Reaction ◽

Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

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Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.

The Journal of Cell Biology ◽

10.1083/jcb.101.2.427 ◽

1985 ◽

Vol 101 (2) ◽

pp. 427-440 ◽

Cited By ~ 40

Author(s):

E Bartnik ◽

M Osborn ◽

K Weber

Keyword(s):

Positive Reaction ◽

Intermediate Filaments ◽

Biochemical Characterization ◽

Helix Pomatia ◽

Molar Ratio ◽

Negative Stain ◽

Epithelial Morphology ◽

Lower Chordate

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.

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Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

Biochemical Journal ◽

10.1042/bj20102131 ◽

2011 ◽

Vol 435 (3) ◽

pp. 733-742 ◽

Cited By ~ 20

Author(s):

Pitter F. Huesgen ◽

Helder Miranda ◽

XuanTam Lam ◽

Manuela Perthold ◽

Holger Schuhmann ◽

...

Keyword(s):

Serine Proteases ◽

Protein Quality ◽

Biochemical Characterization ◽

Pdz Domain ◽

Control Mechanisms ◽

Ph Optimum ◽

Periplasmic Proteins ◽

Synechocystis Sp ◽

Pcc 6803

Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803.

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In Vitro and In Vivo Activities of DA-7867, a New Oxazolidinone, against Aerobic Gram-Positive Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2498-2500.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2498-2500 ◽

Cited By ~ 7

Author(s):

Eun Jeong Yoon ◽

Yeong Woo Jo ◽

Sung Hak Choi ◽

Tae Ho Lee ◽

Jae Keol Rhee ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Streptococcus Pneumoniae ◽

Gram Positive ◽

Methicillin Resistant ◽

Gram Positive Bacteria ◽

Vancomycin Resistant Enterococci ◽

Infection Models ◽

Vancomycin Resistant

ABSTRACT In vitro and in vivo activities of DA-7867 were assessed against methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and penicillin-resistant Streptococcus pneumoniae. All isolates were inhibited by DA-7867 at ≤0.78 μg/ml, a four-times-lower concentration than that of inhibition by linezolid. For murine infection models, DA-7867 also exhibited greater efficacy than linezolid against all isolates tested.

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