enzyme structure
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2021 ◽  
pp. 131877
Author(s):  
Mozhgan Mohammadi ◽  
Behzad Shareghi ◽  
Sadegh Farhadian ◽  
Lida Momeni ◽  
Ali Akbar Saboury

Author(s):  
Andres F. Chaparro Sosa ◽  
Riley M. Bednar ◽  
Ryan A. Mehl ◽  
Daniel K. Schwartz ◽  
Joel L. Kaar

2021 ◽  
Vol 22 (6) ◽  
pp. 3266
Author(s):  
Justin Foster ◽  
Ninghui Cheng ◽  
Vincent Paris ◽  
Lingfei Wang ◽  
Jin Wang ◽  
...  

Considering the widespread occurrence of oxalate in nature and its broad impact on a host of organisms, it is surprising that so little is known about the turnover of this important acid. In plants, oxalate oxidase is the most well-studied enzyme capable of degrading oxalate, but not all plants possess this activity. Recently, acyl-activating enzyme 3 (AAE3), encoding an oxalyl-CoA synthetase, was identified in Arabidopsis. This enzyme has been proposed to catalyze the first step in an alternative pathway of oxalate degradation. Since this initial discovery, this enzyme and proposed pathway have been found to be important to other plants and yeast as well. In this study, we identify, in Arabidopsis, an oxalyl-CoA decarboxylase (AtOXC) that is capable of catalyzing the second step in this proposed pathway of oxalate catabolism. This enzyme breaks down oxalyl-CoA, the product of AtAAE3, into formyl-CoA and CO2. AtOXC:GFP localization suggested that this enzyme functions within the cytosol of the cell. An Atoxc knock-down mutant showed a reduction in the ability to degrade oxalate into CO2. This reduction in AtOXC activity resulted in an increase in the accumulation of oxalate and the enzyme substrate, oxalyl-CoA. Size exclusion studies suggest that the enzyme functions as a dimer. Computer modeling of the AtOXC enzyme structure identified amino acids of predicted importance in co-factor binding and catalysis. Overall, these results suggest that AtOXC catalyzes the second step in this alternative pathway of oxalate catabolism.


2021 ◽  
Author(s):  
Michael Araujo ◽  
Alexandra A. Barrere ◽  
Selena-Rae Tirado ◽  
Candace E. Williams ◽  
Monica I. Strada ◽  
...  

Using crystal structure data, site directed mutagenesis, and real-time kinetic assays, students designed, expressed, and purified engineered mutants of human insulin-degrading enzyme (IDE) to explore structural requirements for enzyme function. Students demonstrated mastery of critical concepts in enzymology including principles of experimental design, implications of enzyme structure-function relationships for molecular evolution, experimental methods for analysis of macromolecular interactions, and computational approaches to scientific inquiry. This investigation was conducted as part of the second-semester undergraduate biochemistry laboratory at Sacred Heart University


2020 ◽  
Vol 48 (10) ◽  
pp. 5603-5615
Author(s):  
Mihaela-Carmen Unciuleac ◽  
Yehuda Goldgur ◽  
Stewart Shuman

Abstract Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3′-OH RNA strands in the context of 3′-OH/5′-PO4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl–AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn2+)2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn2+. The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.


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