The Dual Carboxymethyl Cellulase and Gelatinase Activities of a Newly Isolated Protein from Brevibacillus agri ST15c10 Confer Reciprocal Regulations in Substrate Utilization

2017 ◽  
Vol 27 (6) ◽  
pp. 319-331 ◽  
Author(s):  
Smarajit Maiti ◽  
Tanmoy Samanta ◽  
Sumit Sahoo ◽  
Sudipta Roy
Keyword(s):  
Metal Binding ◽  
Peptidase Activity ◽  
Active Site Residues ◽  
Electron Microscopic ◽  
Endoglucanase Activity ◽  
Significant Similarity ◽  
Gelatinase Activity ◽  
Carboxymethyl Cellulase ◽  
Cmcase Activity ◽  
Brevibacillus Agri

A protein showing endoglucanase-peptidase activity was prepared from a newly isolated bacterium (ST15c10). We identified ST15c10 as <i>Brevibacillus agri</i> based on electron-microscopic images and its 16S-rDNA sequence (GenBank accession No. HM446043), which exhibits 98.9% sequence identity to <i>B. agri </i>(KZ17)/<i>B. formosus </i>(DSM-9885T)/<i>B. brevis</i>. The enzyme was purified to homogeneity and gave a single peak during high-performance liquid chromatography on a Seralose 6B-150 gel-matrix/C-18 column. MALDI-TOF mass-spectrometry and bioinformatics studies revealed significant similarity to M42-aminopeptidases/endoglucanases of the CelM family. These enzymes are found in all <i>Brevibacillus</i> strains for which the genome sequence is known. ST15c10 grows optimally on carboxymethyl cellulose (CMC)-gelatin (40°C/pH 8-9), and also shows strong growth/carboxymethyl cellulase (CMCase) activity in submerged bagasse fermentation. The purified enzyme also functions as endoglucanase with solid bagasse/rice straw. Its CMCase activity (optimal at pH 5.6 and 60°C/K<sub>m</sub> = 35.5 µ<smlcap>M</smlcap>/V<sub>max</sub> = 1,024U) was visualized by zymography on a CMC-polyacrylamide gel, which provided a strong band of approximately 70 kDa. The purified enzyme also showed strong peptidase (gelatinase) activity (pH 7.2/40°C during zymography on 6-12% gelatin/1% gelatin-PAGE (at approx. 70 kDa). The CMCase activity is inhibited by the metal ions Mn/Cu/Fe/Co (50%), Hg/KMnO<sub>4</sub> (100%), and by glucose or lactose (50-75%; all at 1 m<smlcap>M</smlcap>). The observed dose/time-dependent inhibition by Hg ions could be prevented with 2-mercaptoethanol. A comparison of the <i>B. agri</i> endoglucanase-aminopeptidase (ELK43520; 350 aa) with other members of the M42-family revealed the conservation of active-site residues Cys256/Cys260, which were previously identified as metal-binding sites. Regulation of the endoglucanase activity probably occurs via metal binding-triggered changes in the redox state of the enzyme. Studies on this type of enzyme are of high importance for basic scientific and industrial research.

scholarly journals Crystal structure of a pyridoxal 5′-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii

2017 ◽  
Vol 73 (12) ◽  
pp. 651-656 ◽  
Author(s):  
Taichi Mizobuchi ◽  
Risako Nonaka ◽  
Motoki Yoshimura ◽  
Katsumasa Abe ◽  
Shouji Takahashi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Active Site ◽  
Metal Binding ◽  
Bivalve Mollusc ◽  
Active Site Residues ◽  
X Ray ◽  
Aspartate Racemase ◽  
Scapharca Broughtonii

Aspartate racemase (AspR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the β-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

scholarly journals Roles of Active Site Residues and the HUH Motif of the F Plasmid TraI Relaxase

2007 ◽  
Vol 282 (46) ◽  
pp. 33707-33713 ◽  
Author(s):  
Christopher Larkin ◽  
Rembrandt J. F. Haft ◽  
Matthew J. Harley ◽  
Beth Traxler ◽  
Joel F. Schildbach
Keyword(s):  
Metal Binding ◽  
Conjugative Plasmid ◽  
Active Site Residues ◽  
F Plasmid ◽  
Binding Properties ◽  
Single Stranded Dna ◽  
Metal Affinity ◽  
Conjugation Transfer

Bacterial conjugation, transfer of a single strand of a conjugative plasmid between bacteria, requires sequence-specific single-stranded DNA endonucleases called relaxases or nickases. Relaxases contain an HUH (His-hydrophobe-His) motif, part of a three-His cluster that binds a divalent cation required for the cleavage reaction. Crystal structures of the F plasmid TraI relaxase domain, with and without bound single-stranded DNA, revealed an extensive network of interactions involving HUH and other residues. Here we study the roles of these residues in TraI function. Whereas substitutions for the three His residues alter metal-binding properties of the protein, the same substitution at each position elicits different effects, indicating that the residues contribute asymmetrically to metal binding. Substitutions for a conserved Asp that interacts with one HUH His demonstrate that the Asp modulates metal affinity despite its distance from the metal. The bound metal enhances binding of ssDNA to the protein, consistent with a role for the metal in positioning the scissile phosphate for cleavage. Most substitutions tested caused significantly reduced in vitro cleavage activities and in vivo transfer efficiencies. In summary, the results suggest that the metal-binding His cluster in TraI is a finely tuned structure that achieves a sufficient affinity for metal while avoiding the unfavorable electrostatics that would result from placing an acidic residue near the scissile phosphate of the bound ssDNA.

scholarly journals The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.

1989 ◽  
Vol 109 (6) ◽  
pp. 3039-3052 ◽  
Author(s):  
G A Oyler ◽  
G A Higgins ◽  
R A Hart ◽  
E Battenberg ◽  
M Billingsley ◽  
...  
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Granule Cells ◽  
Molecular Layer ◽  
Synaptic Function ◽  
Differentially Expressed ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Thalamic Nuclei ◽  
Electron Microscopic

cDNA clones of a neuronal-specific mRNA encoding a novel 25-kD synaptosomal protein, SNAP-25, that is widely, but differentially expressed by diverse neuronal subpopulations of the mammalian nervous system have been isolated and characterized. The sequence of the SNAP-25 cDNA revealed a single open reading frame that encodes a primary translation product of 206 amino acids. Antisera elicited against a 12-amino acid peptide, corresponding to the carboxy-terminal residues of the predicted polypeptide sequence, recognized a single 25-kD protein that is associated with synaptosomal fractions of hippocampal preparations. The SNAP-25 polypeptide remains associated with synaptosomal membrane components after hypoosmotic lysis and is released by nonionic detergent but not high salt extraction. Although the SNAP-25 polypeptide lacks a hydrophobic stretch of residues compatible with a transmembrane region, the amino terminus may form an amphiphilic helix that may facilitate alignment with membranes. The predicted amino acid sequence also includes a cluster of four closely spaced cysteine residues, similar to the metal binding domains of some metalloproteins, suggesting that the SNAP-25 polypeptide may have the potential to coordinately bind metal ions. Consistent with the protein fractionation, light and electron microscopic immunocytochemistry indicated that SNAP-25 is located within the presynaptic terminals of hippocampal mossy fibers and the inner molecular layer of the dentate gyrus. The mRNA was found to be enriched within neurons of the neocortex, hippocampus, piriform cortex, anterior thalamic nuclei, pontine nuclei, and granule cells of the cerebellum. The distribution of the SNAP-25 mRNA and the association of the protein with presynaptic elements suggest that SNAP-25 may play an important role in the synaptic function of specific neuronal systems.

scholarly journals Insight into the Function of Active Site Residues in the Catalytic Mechanism of Human Ferrochelatase

2021 ◽  
Author(s):  
Amy E. Medlock ◽  
Wided Najahi-Missaoui ◽  
Mesafint T. Shiferaw ◽  
Angela N. Albetel ◽  
William N. Lanzilotta ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Structural Data ◽  
Active Site Residues ◽  
Proton Abstraction ◽  
Product Release ◽  
Iron Delivery ◽  
High Level

Ferrochelatase catalyzes the insertion of ferrous iron into a porphyrin macrocycle to produce the essential cofactor, heme. In humans this enzyme not only catalyzes the terminal step, but also serves a regulatory step in the heme synthesis pathway. Over a dozen crystal structures of human ferrochelatase have been solved and many variants have been characterized kinetically. In addition, hydrogen deuterium exchange, resonance Raman, molecular dynamics, and high level quantum mechanic studies have added to our understanding of  the catalytic cycle of the enzyme. However, an understanding of how the metal ion is delivered and the specific role that active site residues play in catalysis remain open questions. Data are consistent with metal binding and insertion occurring from the side opposite from where pyrrole proton abstraction takes place. To better understand iron delivery and binding as well as the role of conserved residues in the active site, we have constructed and characterized a series of enzyme variants. Crystallographic studies as well as rescue and kinetic analysis of variants were performed. Data from these studies are consistent with the M76 residue playing a role in active site metal binding and formation of a weak iron protein ligand being necessary for product release. Additionally, structural data support a role for E343 in proton abstraction and product release in coordination with a peptide loop composed of Q302, S303 and K304 that act a metal sensor.

scholarly journals Mutational and Metal Binding Analysis of the Endonuclease Domain of the Influenza Virus Polymerase PA Subunit

2010 ◽  
Vol 84 (18) ◽  
pp. 9096-9104 ◽  
Author(s):  
Thibaut Crépin ◽  
Alexandre Dias ◽  
Andrés Palencia ◽  
Christopher Swale ◽  
Stephen Cusack ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Active Site ◽  
Metal Binding ◽  
Specific Binding ◽  
Single Point ◽  
Endonuclease Activity ◽  
Divalent Metal Ions ◽  
Active Site Residues ◽  
Knock Out ◽  
Endonuclease Domain

ABSTRACT Influenza virus polymerase initiates the biosynthesis of its own mRNAs with capped 10- to 13-nucleotide fragments cleaved from cellular (pre-)mRNAs. Two activities are required for this cap-snatching activity: specific binding of the cap structure and an endonuclease activity. Recent work has shown that the cap-binding site is situated in the central part of the PB2 subunit and that the endonuclease activity is situated in the N-terminal domain of the PA subunit (PA-Nter). The influenza endonuclease is a member of the PD-(D/E)XK family of nucleases that use divalent metal ions for nucleic acid cleavage. Here we analyze the metal binding and endonuclease activities of eight PA-Nter single-point mutants. We show by calorimetry that the wild-type active site binds two Mn2+ ions and has a 500-fold higher affinity for manganese than for magnesium ions. The endonuclease activity of the isolated mutant domains are compared with the cap-dependent transcription activities of identical mutations in trimeric recombinant polymerases previously described by other groups. Mutations that inactivate the endonuclease activity in the isolated PA-Nter knock out the transcription but not replication activity in the recombinant polymerase. We confirm the importance of a number of active-site residues and identify some residues that may be involved in the positioning of the RNA substrate in the active site. Our results validate the use of the isolated endonuclease domain in a drug-design process for new anti-influenza virus compounds.

scholarly journals Identification and Molecular Analysis of PcsB, a Protein Required for Cell Wall Separation of Group B Streptococcus

2001 ◽  
Vol 183 (4) ◽  
pp. 1175-1183 ◽  
Author(s):  
Dieter J. Reinscheid ◽  
Birgit Gottschalk ◽  
Axel Schubert ◽  
Bernhard J. Eikmanns ◽  
Gursharan S. Chhatwal
Keyword(s):  
Single Cells ◽  
Group B Streptococcus ◽  
Microscopic Analysis ◽  
Peptide Sequence ◽  
Cosmid Library ◽  
Electron Microscopic ◽  
Significant Similarity ◽  
Signal Peptide Sequence ◽  
Insertional Inactivation ◽  
Group B

ABSTRACT Group B streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis in neonates. N-terminal sequencing of major proteins in the culture supernatant of a clinical isolate of GBS identified a protein of about 50 kDa which could be detected in all of 27 clinical isolates tested. The corresponding gene, designatedpcsB, was isolated from a GBS cosmid library and subsequently sequenced. The deduced PcsB polypeptide consists of 447 amino acid residues (M r, 46,754), carries a potential N-terminal signal peptide sequence of 25 amino acids, and shows significant similarity to open reading frames of unknown function from different organisms and to the murein hydrolase P45 from Listeria monocytogenes. Northern blot analysis revealed a monocistronic transcriptional organization forpcsB in GBS. Insertional inactivation of pcsBin the genome of GBS resulted in mutant strain Sep1 exhibiting a drastically reduced growth rate compared to the parental GBS strain and showing an increased susceptibility to osmotic pressure and to various antibiotics. Electron microscopic analysis of GBS mutant Sep1 revealed growth in clumps, cell separation in several planes, and multiple division septa within single cells. These data suggest a pivotal role of PcsB for cell division and antibiotic tolerance of GBS.

scholarly journals Identification of a calicivirus isolate of unknown origin

2003 ◽  
Vol 84 (10) ◽  
pp. 2837-2845 ◽  
Author(s):  
Angelika Oehmig ◽  
Mathias Büttner ◽  
Frank Weiland ◽  
William Werz ◽  
Klaus Bergemann ◽  
...  
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Unknown Origin ◽  
Open Reading Frames ◽  
Coding Region ◽  
Electron Microscopic ◽  
Significant Similarity ◽  
Western Blots ◽  
Reading Frame ◽  
A Minor

Chinese hamster ovary (CHO) cells manifesting striking cytopathogenic changes in culture were investigated to determine the causative agent. Electron microscopic analyses revealed viral particles of about 40 nm in diameter, displaying typical calicivirus morphology. To date, this virus, designated isolate 2117, exclusively replicates in CHO cells, achieving only moderate titres. After cloning, the coding region of 7928 nucleotides, the 3′ non-coding region and the poly(A) tail were sequenced. The genome consists of three open reading frames (ORFs), with the first and second ORF having the same reading frame. The overall genomic organization as well as the nucleotide sequence of isolate 2117 is most similar to that of a recently described canine calicivirus, but also shows significant similarity to the sequences of mink calicivirus and other caliciviruses within the genus Vesivirus. In Western blots, using antibodies against the viral protease, a stable, unprocessed 3CD protein of 68 kDa was identified in homogenates of 2117-infected CHO cells. Furthermore, antibodies raised against ORF 3 reacted with the respective protein in 2117-virions, demonstrating that this predicted 9 kDa protein is a minor structural component of the virion. In addition, an RT-PCR assay was established to detect 2117 viral RNA in biological products such as foetal bovine serum, which will aid the discovery of the origin and host of the virus.

Structural insights into chaperone-activity enhancement by a K354E mutation in tomato acidic leucine aminopeptidase

2016 ◽  
Vol 72 (5) ◽  
pp. 694-702 ◽  
Author(s):  
Kevin T. DuPrez ◽  
Melissa A. Scranton ◽  
Linda L. Walling ◽  
Li Fan
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Leucine Aminopeptidase ◽  
Gel Filtration ◽  
Chaperone Activity ◽  
Peptidase Activity ◽  
Wild Type ◽  
Active Site Residues ◽  
In The Wild ◽  
Hydrophobic Areas

Tomato plants express acidic leucine aminopeptidase (LAP-A) in response to various environmental stressors. LAP-A not only functions as a peptidase for diverse peptide substrates, but also displays chaperone activity. A K354E mutation has been shown to abolish the peptidase activity but to enhance the chaperone activity of LAP-A. To better understand this moonlighting function of LAP-A, the crystal structure of the K354E mutant was determined at 2.15 Å resolution. The structure reveals that the K354E mutation destabilizes an active-site loop and causes significant rearrangement of active-site residues, leading to loss of the catalytic metal-ion coordination required for the peptidase activity. Although the mutant was crystallized in the same hexameric form as wild-type LAP-A, gel-filtration chromatography revealed an apparent shift from the hexamer to lower-order oligomers for the K354E mutant, showing a mixture of monomers to trimers in solution. In addition, surface-probing assays indicated that the K354E mutant has more accessible hydrophobic areas than wild-type LAP-A. Consistently, computational thermodynamic estimations of the interfaces between LAP-A monomers suggest that increased exposure of hydrophobic surfaces occurs upon hexamer breakdown. These results suggest that the K354E mutation disrupts the active-site loop, which also contributes to the hexameric assembly, and destabilizes the hexamers, resulting in much greater hydrophobic areas accessible for efficient chaperone activity than in the wild-type LAP-A.

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