The Bacillus subtilis Signaling Protein SpoIVB Defines a New Family of Serine Peptidases

ABSTRACT The protein SpoIVB plays a key role in signaling in the ςK checkpoint of Bacillus subtilis. This regulatory mechanism coordinates late gene expression during development in this organism and we have recently shown SpoIVB to be a serine peptidase. SpoIVB signals by transiting a membrane, undergoing self-cleavage, and then by an unknown mechanism activating a zinc metalloprotease, SpoIVFB, which cleaves pro-ςK to its active form, ςK, in the outer mother cell chamber of the developing cell. In this work we have characterized the serine peptidase domain of SpoIVB. Alignment of SpoIVB with homologues from other spore formers has allowed site-specific mutagenesis of all potential active site residues within the peptidase domain. We have defined the putative catalytic domain of the SpoIVB serine peptidase as a 160-amino-acid residue segment at the carboxyl terminus of the protein. His236 and Ser378 are the most important residues for proteolysis, with Asp363 being the most probable third member of the catalytic triad. In addition, we have shown that mutations at residues Asn290 and His394 lead to delayed signaling in the ςK checkpoint. The active site residues suggest that SpoIVB and its homologues from other spore formers are members of a new family of serine peptidases of the trypsin superfamily.

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Activation and selectivity of OTUB-1 and OTUB-2 deubiquitinylases

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013073 ◽

2020 ◽

Vol 295 (20) ◽

pp. 6972-6982

Author(s):

Dakshinamurthy Sivakumar ◽

Vikash Kumar ◽

Michael Naumann ◽

Matthias Stein

Keyword(s):

Active Site ◽

Electrostatic Interactions ◽

Active Form ◽

Cysteine Proteases ◽

Binding Pocket ◽

Catalytic Triad ◽

Catalytic Site ◽

Dynamics Simulation ◽

Active Site Residues ◽

Catalytically Active

The ovarian tumor domain (OTU) deubiquitinylating cysteine proteases OTUB1 and OTUB2 (OTU ubiquitin aldehyde binding 1 and 2) are representative members of the OTU subfamily of deubiquitinylases. Deubiquitinylation critically regulates a multitude of important cellular processes, such as apoptosis, cell signaling, and growth. Moreover, elevated OTUB expression has been observed in various cancers, including glioma, endometrial cancer, ovarian cancer, and breast cancer. Here, using molecular dynamics simulation approaches, we found that both OTUB1 and OTUB2 display a catalytic triad characteristic of proteases but differ in their configuration and protonation states. The OTUB1 protein had a prearranged catalytic site, with strong electrostatic interactions between the active-site residues His265 and Asp267. In OTUB2, however, the arrangement of the catalytic triad was different. In the absence of ubiquitin, the neutral states of the catalytic-site residues in OTUB2 were more stable, resulting in larger distances between these residues. Only upon ubiquitin binding did the catalytic triad in OTUB2 rearrange and bring the active site into a catalytically feasible state. An analysis of water access channels revealed only a few diffusion trajectories for the catalytically active form of OTUB1, whereas in OTUB2 the catalytic site was solvent-accessible, and a larger number of water molecules reached and left the binding pocket. Interestingly, in OTUB2, the catalytic residues His224 and Asn226 formed a stable hydrogen bond. We propose that the observed differences in activation kinetics, protonation states, water channels, and active-site accessibility between OTUB1 and OTUB2 may be relevant for the selective design of OTU inhibitors.

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Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis

The Journal of Biochemistry ◽

10.1093/jb/mvab135 ◽

2021 ◽

Author(s):

Ryuji Yamazawa ◽

Ritsuko Kuwana ◽

Kenji Takeuchi ◽

Hiromu Takamatsu ◽

Yoshitaka Nakajima ◽

...

Keyword(s):

Bacillus Subtilis ◽

Chemical Modification ◽

Cysteine Protease ◽

Active Site ◽

Amino Acid Sequences ◽

Site Directed Mutagenesis ◽

Protease Gene ◽

Active Site Residues ◽

Protein Determination ◽

35 Kda Protein

Abstract In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) in Escherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulfhydryl groups exerted significant effects, strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibited the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the Merops peptidase database.

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The Herpesvirus Proteases as Targets for Antiviral Chemotherapy

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020001100101 ◽

2000 ◽

Vol 11 (1) ◽

pp. 1-22 ◽

Cited By ~ 85

Author(s):

Lloyd Waxman ◽

Paul L Darke

Keyword(s):

Serine Protease ◽

Active Site ◽

Serine Proteases ◽

Catalytic Triad ◽

Active Site Residues ◽

Virus Family ◽

Single Domain Protein ◽

Inhibitor Discovery ◽

Antiviral Chemotherapy ◽

Simplex Virus

Viruses of the family Herpesviridae are responsible for a diverse set of human diseases. The available treatments are largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. For several members of this DNA virus family, advances have been made recently in the biochemistry and structural biology of the essential viral protease, revealing common features that may be possible to exploit in the development of a new class of anti-herpesvirus agents. The herpesvirus proteases have been identified as belonging to a unique class of serine protease, with a Ser-His-His catalytic triad. A new, single domain protein fold has been determined by X-ray crystallography for the proteases of at least three different herpesviruses. Also unique for serine proteases, dimerization has been shown to be required for activity of the cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods needed for productive, functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus proteases lead to common considerations for this group of proteases in the early phases of inhibitor discovery. In general, classical serine protease inhibitors that react with active site residues do not readily inactivate the herpesvirus proteases. There has been progress however, with activated carbonyls that exploit the selective nucleophilicity of the active site serine. In addition, screening of chemical libraries has yielded novel structures as starting points for drug development. Recent crystal structures of the herpesvirus proteases now allow more direct interpretation of ligand structure—activity relationships. This review first describes basic functional aspects of herpesvirus protease biology and enzymology. Then we discuss inhibitors identified to date and the prospects for their future development.

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Identification of Essential Residues in Apolipoprotein N-Acyl Transferase, a Member of the CN Hydrolase Family

Journal of Bacteriology ◽

10.1128/jb.00099-07 ◽

2007 ◽

Vol 189 (12) ◽

pp. 4456-4464 ◽

Cited By ~ 33

Author(s):

Dominique Vidal-Ingigliardi ◽

Shawn Lewenza ◽

Nienke Buddelmeijer

Keyword(s):

Active Site ◽

Structural Model ◽

Protein Structures ◽

Catalytic Triad ◽

Site Directed Mutagenesis ◽

Acyl Transferase ◽

Active Site Residues ◽

Flexible Arms ◽

Hydrolase Family

ABSTRACT Apolipoprotein N-acyl transferase (Lnt) is an essential membrane-bound protein involved in lipid modification of all lipoproteins in gram-negative bacteria. Essential residues in Lnt of Escherichia coli were identified by using site-directed mutagenesis and an in vivo complementation assay. Based on sequence conservation and known protein structures, we predict a model for Lnt, which is a member of the CN hydrolase family. Besides the potential catalytic triad E267-K335-C387, four residues that directly affect the modification of Braun's lipoprotein Lpp are absolutely required for Lnt function. Residues Y388 and E389 are part of the hydrophobic pocket that constitutes the active site. Residues W237 and E343 are located on two flexible arms that face away from the active site and are expected to open and close upon the binding and release of phospholipid and/or apolipoprotein. Substitutions causing temperature-dependent effects were located at different positions in the structural model. These mutants were not affected in protein stability. Lnt proteins from other proteobacteria, but not from actinomycetes, were functional in vivo, and the essential residues identified in Lnt of E. coli are conserved in these proteins.

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Site-directed mutagenesis of active site residues in Bacillus subtilis α-amylase

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/0167-4838(92)90249-d ◽

1992 ◽

Vol 1120 (3) ◽

pp. 281-288 ◽

Cited By ~ 42

Author(s):

Kenji Takase ◽

Takashi Matsumoto ◽

Hiroshi Mizuno ◽

Kazio Yamane

Keyword(s):

Bacillus Subtilis ◽

Active Site ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Active Site Residues

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Biological and Structural Characterization of Trypanosoma cruzi Phosphodiesterase C and Implications for Design of Parasite Selective Inhibitors

Journal of Biological Chemistry ◽

10.1074/jbc.m111.326777 ◽

2012 ◽

Vol 287 (15) ◽

pp. 11788-11797 ◽

Cited By ~ 26

Author(s):

Huanchen Wang ◽

Stefan Kunz ◽

Gong Chen ◽

Thomas Seebeck ◽

Yiqian Wan ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Crystal Structures ◽

Active Site ◽

Catalytic Domain ◽

Trypanosoma Evansi ◽

Active Site Residues ◽

Dual Specificity ◽

Pde Inhibitors ◽

Starting Model

Trypanosoma cruzi phosphodiesterase C (TcrPDEC) is a potential new drug target for the treatment of Chagas disease but has not been well studied. This study reports the enzymatic properties of various kinetoplastid PDECs and the crystal structures of the unliganded TcrPDEC1 catalytic domain and its complex with an inhibitor. Mutations of PDEC during the course of evolution led to inactivation of PDEC in Trypanosoma brucei/Trypanosoma evansi/Trypanosoma congolense, whereas the enzyme is active in all other kinetoplastids. The TcrPDEC1 catalytic domain hydrolyzes both cAMP and cGMP with a Km of 23.8 μm and a kcat of 31 s−1 for cAMP and a Km of 99.1 μm and a kcat of 17 s−1 for cGMP, thus confirming its dual specificity. The crystal structures show that the N-terminal fragment wraps around the TcrPDEC catalytic domain and may thus regulate its enzymatic activity via direct interactions with the active site residues. A PDE5 selective inhibitor that has an IC50 of 230 nm for TcrPDEC1 binds to TcrPDEC1 in an orientation opposite to that of sildenafil. This observation, together with the screen of the inhibitory potency of human PDE inhibitors against TcrPDEC, implies that the scaffold of some human PDE inhibitors might be used as the starting model for design of parasite PDE inhibitors. The structural study also identified a unique parasite pocket that neighbors the active site and may thus be valuable for the design of parasite-specific inhibitors.

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Mutational Analysis of Active Site Residues Essential for Sensing of Organic Hydroperoxides by Bacillus subtilis OhrR

Journal of Bacteriology ◽

10.1128/jb.00879-07 ◽

2007 ◽

Vol 189 (19) ◽

pp. 7069-7076 ◽

Cited By ~ 16

Author(s):

Sumarin Soonsanga ◽

Mayuree Fuangthong ◽

John D. Helmann

Keyword(s):

Hydrogen Bond ◽

Bacillus Subtilis ◽

Conformational Changes ◽

Active Site ◽

Mutational Analysis ◽

High Sensitivity ◽

Oxidative Modification ◽

Active Site Residues

ABSTRACT Bacillus subtilis OhrR is the prototype for the one-Cys family of organic peroxide-sensing regulatory proteins. Mutational analyses indicate that the high sensitivity of the active site cysteine (C15) to peroxidation requires three Tyr residues. Y29 and Y40 from the opposing subunit of the functional dimer hydrogen bond with the reactive Cys thiolate, and substitutions at these positions reduce or eliminate the ability of OhrR to respond to organic peroxides. Y19 is also critical for peroxide sensing, and the Ala substitution mutant (OhrR Y19A) is less susceptible to oxidation at the active site C15 in vivo. The Y19A protein also displays decreased sensitivity to peroxide-mediated oxidation in vitro. Y19 is in van der Waals contact with two residues critical for protein function, F16 and R23. The latter residue makes critical contact with the DNA backbone in the OhrR-operator complex. These results indicate that the high sensitivity of the OhrR C15 residue to oxidation requires interactions with the opposed Tyr residues. Oxidative modification of C15 likely disrupts the C15-Y29′-Y40′ hydrogen bond network and thereby initiates conformational changes that reduce the ability of OhrR to bind to its operator site.

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Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

Infection and Immunity ◽

10.1128/iai.66.5.2374-2378.1998 ◽

1998 ◽

Vol 66 (5) ◽

pp. 2374-2378 ◽

Cited By ~ 54

Author(s):

S. E. Hammond ◽

P. C. Hanna

Keyword(s):

Active Site ◽

Lethal Factor ◽

Peptide Sequencing ◽

The Novel ◽

Active Site Residues ◽

Zinc Metalloprotease ◽

Proteolytic Activities ◽

Substitution Mutations ◽

Catalytic Functions

ABSTRACT The lethal factor (LF) protein of Bacillus anthracislethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase.

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Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop

Biochemical Journal ◽

10.1042/bj3560327 ◽

2001 ◽

Vol 356 (2) ◽

pp. 327-334 ◽

Cited By ~ 20

Author(s):

Fernando RODRIGUES-LIMA ◽

Claudine DELOMÉNIE ◽

Geoffrey H. GOODFELLOW ◽

Denis M. GRANT ◽

Jean-Marie DUPRET

Keyword(s):

Crystal Structure ◽

Cysteine Protease ◽

Active Site ◽

Catalytic Mechanism ◽

Catalytic Domain ◽

Homology Modelling ◽

Metabolic Activation ◽

Cysteine Proteases ◽

Catalytic Triad ◽

Quality Model

Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560–564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29–131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122–131 in NAT1 model and residues 122–133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.

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