Properties of Inosinic Acid Dehydrogenase from Bacillus subtilis. I. Purification and Physical Properties

1973 ◽  
Vol 51 (10) ◽  
pp. 1380-1390 ◽  
Author(s):  
Tai-Wing Wu ◽  
K. G. Scrimgeour
Keyword(s):  
Bacillus Subtilis ◽  
Quaternary Structure ◽  
Active Form ◽  
Kinetic Properties ◽  
Purification Method ◽  
Molecular Weights ◽  
Inosinic Acid ◽  
Apparent Homogeneity ◽  
Catalytically Active ◽  
Membrane Bound

IMP (inosinic acid or inosine-5′-phosphate) dehydrogenase has been purified to apparent homogeneity from Bacillus subtilis. The purification method yields an enzyme preparation that retains a constant level of inhibition by guanosine 5′-phosphate. The enzyme is membrane bound, and can be removed from membrane material after treatment either with detergents or with phospholipase A. Both the membrane-bound and solubilized forms of IMP dehydrogenase have similar kinetic properties. The soluble enzyme can occur in a number of oligomeric forms, with molecular weights that are multiples of 100 000 daltons. Although both the tetramer and the dimer appear to be catalytically active, no conclusions can yet be drawn about the quaternary structure of the enzymically active form(s).

scholarly journals In vitro characterization of the colibactin-activating peptidase ClbP enables development of a fluorogenic activity probe

2019 ◽  
Author(s):  
Matthew R. Volpe ◽  
Matthew R. Wilson ◽  
Carolyn A. Brotherton ◽  
Ethan S. Winter ◽  
Sheila E. Johnson ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Biochemical Characterization ◽  
Active Form ◽  
Inner Membrane ◽  
Substrate Preferences ◽  
In Vitro Characterization ◽  
Catalytically Active ◽  
Membrane Bound

ABSTRACTThe gut bacterial genotoxin colibactin has been linked to the development of colorectal cancer. In the final stages of colibactin’s biosynthesis, an inactive precursor (pre-colibactin) undergoes proteolytic cleavage by ClbP, an unusual inner-membrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studied in vitro and limited tools exist to measure its activity. Here, we describe the in vitro biochemical characterization of catalytically active, full-length ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactin-producing bacteria.

Properties of Inosinic Acid Dehydrogenase from Bacillus subtilis. II. Kinetic Properties

1973 ◽  
Vol 51 (10) ◽  
pp. 1391-1398 ◽  
Author(s):  
Tai-Wing Wu ◽  
K. G. Scrimgeour
Keyword(s):  
Bacillus Subtilis ◽  
Kinetic Properties ◽  
Ph Optimum ◽  
Ionic Radii ◽  
Imp Dehydrogenase ◽  
Inosinic Acid ◽  
Feedback Inhibitor ◽  
Enzyme Change ◽  
A Site ◽  
Complex Manner

Some of the kinetic properties of inosine 5′-phosphate (IMP) dehydrogenase purified from Bacillus subtilis have been described. K+ can be replaced as an activator by other monovalent cations, whose activities are related to their ionic radii. IMP dehydrogenase is stimulated by appropriate concentrations of adenosine 5′-phosphate, and is strongly inhibited by the antibiotic mycophenolic acid. The enzyme is inhibited by guanosine 5′-phosphate (GMP), a suspected negative feedback inhibitor, in a complex manner. Desensitization of this inhibition suggests that GMP binds at a site distinct from the catalytic site. Several properties of the enzyme change after desensitization. For example, the pH optimum shifts from pH 7.55 to pH 8.4, and whereas the inhibition by GMP is greatly reduced there is an increase in catalytic activity. These studies and the oligomeric structure of IMP dehydrogenase strongly indicate a regulatory role for the enzyme in B. subtilis.

scholarly journals Engineering subtilisin proteases that specifically degrade active RAS

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yingwei Chen ◽  
Eric A. Toth ◽  
Biao Ruan ◽  
Eun Jung Choi ◽  
Richard Simmerman ◽  
...  
Keyword(s):  
Active Form ◽  
High Specificity ◽  
Kinetic Properties ◽  
Target Sequence ◽  
Reporter System ◽  
Human Cell Culture ◽  
Conserved Sequence ◽  
Cofactor Binding ◽  
Selective Proteolysis

AbstractWe describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

scholarly journals Topology and quaternary structure of pro-sucrase/isomaltase and final-form sucrase/isomaltase

1986 ◽  
Vol 237 (2) ◽  
pp. 455-461 ◽  
Author(s):  
G M Cowell ◽  
J Tranum-Jensen ◽  
H Sjöström ◽  
O Norén
Keyword(s):  
Electron Microscopy ◽  
Quaternary Structure ◽  
Functional Organization ◽  
Enzyme Complex ◽  
Single Chain ◽  
Pancreatic Duct Ligation ◽  
Duct Ligation ◽  
Catalytically Active ◽  
Identical Series ◽  
Dimeric Model

Pig sucrase/isomaltase (EC 3.2.1.48/10) was purified from intestinal microvillar vesicles prepared from animals with and without pancreatic-duct ligation to obtain the single-chain pro form and the proteolytically cleaved final form respectively. The purified enzymes were re-incorporated into phosphatidylcholine vesicles and analysed by electron microscopy after negative staining. The two forms of the enzyme were observed as identical series of characteristic projected views that could be unified in a single dimeric model, containing two sucrase and two isomaltase units. This shows a homodimeric functional organization similar to that of other microvillar hydrolases. The bulk of the dimer was separated from the membrane by a maximal gap of 3.5 nm, representing a junctional segment connecting the intramembrane section of the anchor to the catalytically active domain of sucrase/isomaltase. The enzyme complex protrudes from the membrane for a distance of up to 17 nm. From charge-shift immunoelectrophoresic studies of hydrophilic prosucrase/isomaltase and from electron microscopy of reconstituted pro-sucrase/isomaltase, there was no evidence to suggest the presence of anchoring sequences between the sucrase and isomaltase subunits.

Tetramer formation of Bacillus subtilis YabJ protein that belongs to YjgF/YER057c/UK114 family

2021 ◽  
Vol 85 (2) ◽  
pp. 297-306
Author(s):  
Zui Fujimoto ◽  
Le Thi Thu Hong ◽  
Naomi Kishine ◽  
Nobuhiro Suzuki ◽  
Keitarou Kimura
Keyword(s):  
Bacillus Subtilis ◽  
Quaternary Structure ◽  
Single Amino Acid ◽  
Wild Type ◽  
Amino Acid Mutation ◽  
X Ray Crystallography ◽  
Ligand Binding Sites ◽  
Potential Ligand ◽  
Single Amino Acid Mutation

ABSTRACT Bacillus subtilis YabJ protein belongs to the highly conserved YjgF/YER057c/UK114 family, which has a homotrimeric quaternary structure. The dominant allele of yabJ gene that is caused by a single amino acid mutation of Ser103Phe enables poly-γ-glutamic acid (γPGA) production of B. subtilis under conditions where the cell-density signal transduction was disturbed by the loss of DegQ function. X-ray crystallography of recombinant proteins revealed that unlike the homotrimeric wild-type YabJ, the mutant YabJ(Ser103Phe) had a homotetrameric quaternary structure, and the structural change appeared to be triggered by an inversion of the fifth β-strand. The YabJ homotetramer has a hole that is highly accessible, penetrating through the tetramer, and 2 surface concaves as potential ligand-binding sites. Western blot analyses revealed that the conformational change was also induced in vivo by the Ser103Phe mutation.

Interaction of Mycoplasma gallisepticum with sialyl glycoproteins

1980 ◽  
Vol 30 (2) ◽  
pp. 353-361
Author(s):  
L R Glasgow ◽  
R L Hill
Keyword(s):  
Sialic Acid ◽  
Mycoplasma Gallisepticum ◽  
Binding Assays ◽  
Molecular Weights ◽  
Acetylneuraminic Acid ◽  
Direct Binding ◽  
Membrane Bound ◽  
Strict Specificity ◽  
Human Glycophorin ◽  
Acid Structure

The binding of several glycoproteins to freshly grown and harvested cells of Mycoplasma gallisepticum was examined. Only human glycophorin, the major sialoglycoprotein of the erythrocyte membrane, bound tightly as judged by direct binding assays with 125I-labeled glycoproteins. Neuraminidase-treated glycophorin did not bind, suggesting that binding is mediated through sialic acid groups. Although other sialoglycoproteins did not appear to bind M. gallisepticum by direct binding assays, some inhibited the binding of glycophorin. The best inhibitors had a mucin-like structure, with high molecular weights and high sialic acid contents. N-acetylneuraminic acid appeared to be the favored sialic acid structure for binding, but there was no strict specificity for its anomeric linkage. Neuraminidase activity could not be detected on the surface of M. gallisepticum, suggesting that this enzyme is not involved in the mechanism of adherence of sialoglycoproteins. Binding of sialoglycoproteins was time dependent, however, and markedly diminished with increasing ionic strength, but was largely unaffected between pH 4 and 9.

Heterogeneity of High-molecular-weight Human Salivary Mucins

2000 ◽  
Vol 14 (1) ◽  
pp. 69-75 ◽  
Author(s):  
G.D. Offner ◽  
R.F. Troxler
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Biochemical Properties ◽  
Salivary Proteins ◽  
Molecular Weights ◽  
Structural Framework ◽  
Buccal Epithelial Cells ◽  
Membrane Bound ◽  
Membrane Spanning ◽  
Micro Organisms

The existence of high-molecular-weight glycoproteins in saliva and salivary secretions has been recognized for nearly 30 years. These proteins, called mucins, are essential for oral health and perform many diverse functions in the oral cavity. Mucins have been intensively studied, and much has been learned about their biochemical properties and their interactions with oral micro-organisms and other salivary proteins. In the past several years, the major high-molecular-weight mucin in salivary secretions has been identified as MUC5B, one of a family of 11 human mucin gene products expressed in tissue-specific patterns in the gastrointestinal, respiratory, and reproductive tracts. MUC5B is one of four gel-forming mucins which exist as multimeric proteins with molecular weights greater than 20-40 million daltons. The heavily glycosylated mucin multimers form viscous layers which protect underlying epithelial surfaces from microbial, mechanical, and chemical assault. Another class of mucin molecules, the membrane-bound mucins, is structurally and functionally distinct from the gel-forming mucins. These proteins do not form multimers and can exist as both secreted and membrane-bound forms, with the latter anchored to epithelial cell membranes through a short membrane-spanning domain. In the present work, we show that two of the membrane-bound mucins, MUC1 and MUC4, are expressed in all major human salivary glands as well as in buccal epithelial cells. While the functions of these mucins in the oral environment are not understood, it is possible that they form a structural framework on the cell surface which not only is cytoprotective, but also may serve as a scaffold upon which MUC5B, and possibly other salivary proteins, assemble.

scholarly journals Characterization of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatases

1986 ◽  
Vol 233 (3) ◽  
pp. 839-844 ◽  
Author(s):  
P P J Dunn ◽  
A R Slabas ◽  
A L Moore
Keyword(s):  
Adenosine Triphosphatase ◽  
Kinetic Properties ◽  
Ph Profile ◽  
Enzyme Preparations ◽  
Adenosine Triphosphatases ◽  
Membrane Bound ◽  
Cold Stability ◽  
Arum Maculatum ◽  
Guanosine Triphosphatase ◽  
Soluble Enzymes

The catalytic properties of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatase have been analysed. The pH profile, effect of inhibitors, cold-stability and substrate specificity are characteristic of mitochondrial adenosine triphosphatases, although a high guanosine triphosphatase activity does appear to be restricted to plant mitochondrial adenosine triphosphatases. The kinetic properties of nucleoside 5′-triphosphate hydrolysis by membrane-bound and soluble enzymes have been studied by means of double-reciprocal plots. These plots were linear in the absence of an activating anion, which may indicate that the catalytic and/or regulatory mechanism of Arum maculatum adenosine triphosphatase is different from that of other enzyme preparations. It is suggested that the differences in subunit composition of plant and mammalian adenosine triphosphatases reported previously [Dunn, Slabas & Moore (1985) Biochem. J. 225, 821-824] are structurally, rather than functionally, significant.

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