A STUDY ON KINETIC CHARACTERIZATION OF CHANNEL-BLOCKING MUTANTS IN BRADYRHIZOBIUM JAPONICUM UTILIZATION A

2021 ◽  
pp. 49-52
Author(s):  
Anand Shanker Singh ◽  
G. Radhika ◽  
R. Praveen Kumar ◽  
Debarshi Jana
Keyword(s):  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Kinetic Data ◽  
Active Sites ◽  
Substrate Channeling ◽  
Site Analysis ◽  
Proline Utilization ◽  
Channel Blocking ◽  
Proline Utilization A

Proline utilization A (PutA) from Bradyrhizobium japonicum (BjPutA) is a bifunctional avoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and ∆1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate P5C from the PRODH domain to the P5CDH domain. In this work several mutations were made along the channel in an effort to block passage of P5C to the second active site. Analysis of several site-specic mutants in the substrate channel of BjPutA revealed an important role for D779 in the channeling path. BjPutA mutants D779Y and D779W signicantly decreased the overall PRODH-P5CDH channeling reaction indicating that bulky mutations at residue D779 impede travel of P5C through the channel. Interestingly, D779Y and D779W also exhibited lower P5CDH activity, suggesting that exogenous P5C must enter the channel upstream of D779. Replacing D779 with a smaller residue (D779A) had no effect on the catalytic and channeling properties of BjPutA showing that the carboxylate group of D779 is not essential for channeling. An identical mutation at D778 (D778Y) did not impact BjPutA channeling activity. Thus, D779 is optimally orientated so that replacement with the larger side chains of Tyr/Trp blocks P5C movment through the channel. The kinetic data reveal not only that bulky mutations at residue D779 hinder passage of P5C to the second active site, but also P5C must use the channel to efciently access the P5CDH domain. Moreover, these mutants may be used to learn more about the hydrolysis event that is thought to take place within the channel

scholarly journals Kinetic and Structural Characterization of Tunnel-Perturbing Mutants in Bradyrhizobium japonicum Proline Utilization A

Biochemistry ◽  
2014 ◽  
Vol 53 (31) ◽  
pp. 5150-5161 ◽  
Author(s):  
Benjamin W. Arentson ◽  
Min Luo ◽  
Travis A. Pemberton ◽  
John J. Tanner ◽  
Donald F. Becker
Keyword(s):  
Structural Characterization ◽  
Bradyrhizobium Japonicum ◽  
Proline Utilization ◽  
Proline Utilization A

scholarly journals Multi-active Site Dynamics on a Molecular Cr/Co/Se Cluster Catalyst

2022 ◽  
Author(s):  
Jonathan Kephart ◽  
Benjamin Mitchell ◽  
Werner Kaminsky ◽  
Alexandra Velian
Keyword(s):  
Structural Analysis ◽  
Active Site ◽  
Resting State ◽  
Active Sites ◽  
Stepwise Mechanism ◽  
Nitrene Transfer ◽  
Catalytic Intermediates ◽  
Edge Site ◽  
Catalytically Active

This study provides detailed insights into the interconnected reactivity of the three catalytically active sites of an atomically precise nanocluster Cr3(py)3Co6Se8L6 (Cr3(py)3, L = Ph2PNTol–, Ph = phenyl, Tol = 4-tolyl). Catalytic and stoichiometric studies into tosyl azide activation and carbodiimide formation enabled the isolation and crystallographic characterization of key metal-nitrenoid catalytic intermediates, including the tris(nitrenoid) cluster Cr3(NTs)3, the catalytic resting state Cr3(NTs)3(CNtBu)3, and the mono(nitrenoid) cluster Cr3(NTs)(CNtBu)2. Nitrene transfer proceeds via a stepwise mechanism, with the three active sites engaging sequentially to produce carbodiimide. Comparative structural analysis and CNtBu bind-ing studies reveal that the chemical state of neighboring active sites regulates the affinity for substrates of an individual Cr-nitrenoid edge site, intertwining their reactivity through the inorganic support.

scholarly journals High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

2010 ◽  
Vol 54 (10) ◽  
pp. 4343-4351 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Manuela Benvenuti ◽  
Vito Calderone ◽  
Magdalena Stoczko ◽  
Nicola Menciassi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Active Sites ◽  
Structural Diversity ◽  
Binding Pocket ◽  
Structural Basis ◽  
Side Chain ◽  
Functional Heterogeneity ◽  
Hydrophobic Binding

ABSTRACT Metallo-β-lactamases (MBLs) are important enzymatic factors in resistance to β-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes β-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for β-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.

scholarly journals Structure and characterization of a class 3B proline utilization A: Ligand-induced dimerization and importance of the C-terminal domain for catalysis

2017 ◽  
Vol 292 (23) ◽  
pp. 9652-9665 ◽  
Author(s):  
David A. Korasick ◽  
Thameesha T. Gamage ◽  
Shelbi Christgen ◽  
Kyle M. Stiers ◽  
Lesa J. Beamer ◽  
...  
Keyword(s):  
Terminal Domain ◽  
Proline Utilization ◽  
Proline Utilization A

scholarly journals Characterization and Structure Elucidation of Binary Zr:Ti MEL Structure; Simultaneous Photodegradation/Removal of Organic–Inorganic Pollutants

Catalysts ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 633
Author(s):  
Samia A. Kosa ◽  
Islam H. Abd El Maksod ◽  
Eman Z. Hegazy ◽  
Naha M. Al-sebaii
Keyword(s):  
Crystalline Phase ◽  
Active Site ◽  
Structure Elucidation ◽  
Active Sites ◽  
Mutual Effect ◽  
Blue Dye ◽  
Inorganic Pollutants ◽  
Full Characterization ◽  
Cobalt And Nickel

The preparation of a series of different Ti/Zr MEL structure was performed successfully. Full characterization of the prepared materials was done using XRD, IR, DR, and SEM. The results show that the prepared materials contain only one crystalline phase (ZSM-11). The affinity of Zr to form the crystalline phase alone in a binary Zr/Ti synthesizing mixture was approved by SEM and elemental analysis results. The percentage of each active site was calculated. DR spectra were deconvoluted, and three active sites were supposed and quantified (tetragonal, octahedral, and crystalline). The mutual effect of ions (lead, copper, cobalt, and nickel) and methylene blue dye on the removal efficiency with and without ultraviolet irradiation was examined and fully characterized. The ions largely influence the photodegradation process, and a mechanism was formulated. Meanwhile, the presence of dye showed a negligible effect on the removal of ions.

scholarly journals Characterization of the Interaction between the Nuclease and Reverse Transcriptase Activity of the Yeast Telomerase Complex

2000 ◽  
Vol 20 (18) ◽  
pp. 6806-6815 ◽  
Author(s):  
Hongwu Niu ◽  
Jinqiang Xia ◽  
Neal F. Lue
Keyword(s):  
Reverse Transcriptase ◽  
Active Site ◽  
Active Sites ◽  
Biochemical Properties ◽  
Reverse Transcriptase Activity ◽  
Transcriptase Activity ◽  
Yeast Telomerase ◽  
Enzyme Cleavage ◽  
Enzyme Cleavage Site

ABSTRACT Telomerase is a ribonucleoprotein that mediates extension of the dG-rich strand of telomeres in most eukaryotes. Like telomerase derived from ciliated protozoa, yeast telomerase is found to possess a tightly associated endonuclease activity that copurifies with the polymerization activity over different affinity-chromatographic steps. As is the case for ciliate telomerase, primers containing sequences that are not complementary to the RNA template can be efficiently cleaved by the yeast enzyme. More interestingly, we found that for the yeast enzyme, cleavage site selection is not stringent, since blocking cleavage at one site by the introduction of a nonhydrolyzable linkage can lead to the utilization of other sites. In addition, the reverse transcriptase activity of yeast telomerase can extend either the 5′- or 3′-end fragment following cleavage. Two general models that are consistent with the biochemical properties of the enzyme are presented: one model postulates two distinct active sites for the nuclease and reverse transcriptase, and the other invokes a multimeric enzyme with each protomer containing a single active site capable of mediating both cleavage and extension.

scholarly journals Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum

2010 ◽  
Vol 107 (7) ◽  
pp. 2878-2883 ◽  
Author(s):  
D. Srivastava ◽  
J. P. Schuermann ◽  
T. A. White ◽  
N. Krishnan ◽  
N. Sanyal ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Bradyrhizobium Japonicum ◽  
Proline Utilization ◽  
Proline Utilization A
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