substrate binding sites
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2021 ◽  
Vol 8 (1) ◽  
pp. 37
Author(s):  
Zili Song ◽  
Maoqiang He ◽  
Ruilin Zhao ◽  
Landa Qi ◽  
Guocan Chen ◽  
...  

As an indispensable essential amino acid in the human body, lysine is extremely rich in edible mushrooms. The α-aminoadipic acid (AAA) pathway is regarded as the biosynthetic pathway of lysine in higher fungal species in Agaricomycetes. However, there is no deep understanding about the molecular evolutionary relationship between lysine biosynthesis and species in Agaricomycetes. Herein, we analyzed the molecular evolution of lysine biosynthesis in Agaricomycetes. The phylogenetic relationships of 93 species in 34 families and nine orders in Agaricomycetes were constructed with six sequences of LSU, SSU, ITS (5.8 S), RPB1, RPB2, and EF1-α datasets, and then the phylogeny of enzymes involved in the AAA pathway were analyzed, especially homocitrate synthase (HCS), α-aminoadipate reductase (AAR), and saccharopine dehydrogenase (SDH). We found that the evolution of the AAA pathway of lysine biosynthesis is consistent with the evolution of species at the order level in Agaricomycetes. The conservation of primary, secondary, predicted tertiary structures, and substrate-binding sites of the enzymes of HCS, AAR, and SDH further exhibited the evolutionary conservation of lysine biosynthesis in Agaricomycetes. Our results provide a better understanding of the evolutionary conservation of the AAA pathway of lysine biosynthesis in Agaricomycetes.


Author(s):  
Yue Zhou ◽  
Chelsi D. Cassilly ◽  
Todd B. Reynolds

The fungal phosphatidylserine (PS) synthase, a membrane protein encoded by the CHO1 gene, is a potential drug target for pathogenic fungi, such as Candida albicans. However, both substrate-binding sites of C. albicans Cho1 have not been characterized. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies identified a conserved CDP-alcohol phosphotransferase (CAPT) binding motif, which is present within Cho1. We tested the CAPT motif for its role in PS synthesis by mutating conserved residues using alanine substitution mutagenesis. PS synthase assays revealed that mutations in all but one conserved amino acid within the CAPT motif resulted in decreased Cho1 function. In contrast, there were no clear motifs in Cho1 for binding serine. Therefore, to identify the serine binding site, PS synthase sequences from three fungi were aligned with sequences of a similar enzyme, phosphatidylinositol (PI) synthase, from the same fungi. This revealed a motif that was unique to PS synthases. Using alanine substitution mutagenesis, we found that some of the residues in this motif are required for Cho1 function. Two alanine substitution mutants, L184A and R189A, exhibited contrasting impacts on PS synthase activity, and were characterized for their Michaelis-Menten kinetics. The L184A mutant displayed enhanced PS synthase activity and showed an increased Vmax. In contrast, R189A showed decreased PS synthase activity and increased Km for serine, suggesting that residue R189 is involved in serine binding. These results help to characterize PS synthase substrate binding, and should direct rational approaches for finding Cho1 inhibitors that may lead to better antifungals.


2021 ◽  
Author(s):  
Jie Cui ◽  
Haiqin Chen ◽  
Xin Tang ◽  
Hao Zhang ◽  
YongQ Chen ◽  
...  

Abstract Enzyme catalyzed desaturation of intracellular fatty acids plays an important role in various physiological and pathological processes related to lipids. Limited to the multiple transmembrane domains, it is difficult to obtain their three-dimensional structure of fatty acid desaturases. So how they interact with their substrates is unclear. Here, we predicted the complex of Micromonas pusilla delta 6 desaturase (MpFADS6) with the substrate linoleinyl-CoA (ALA-CoA) by trRosetta software and docking poses by Dock 6 software. The potential enzyme-substrate binding sites were anchored by analysis of the complex. Then, site-directed mutagenesis and activity verification clarified that W290, W224, and F352 were critical residues of the substrate tunnel and directly bonded to ALA-CoA. H94 and H69 were indispensable for transporting electrons with heme. H452, N445, and H358 significantly influenced the recognition and attraction of MpFADS6 to the substrate. These findings provide new insights and methods to determine the structure, mechanisms and directed transformation of membrane-bound desaturases.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thanuja D. Sudasinghe ◽  
Michael T. Banco ◽  
Donald R. Ronning

AbstractErgothioneine (EGT) is a low molecular weight histidine betaine essential in all domains of life but only synthesized by selected few organisms. Synthesis of EGT by Mycobacterium tuberculosis (M. tb) is critical for maintaining bioenergetic homeostasis and protecting the bacterium from alkylating agents, oxidative stress, and anti-tubercular drugs. EgtD, an S-adenosylmethionine-dependent methyltransferase (AdoMet), catalyzes the trimethylation of L-Histidine to initiate EGT biosynthesis and this reaction has been shown to be essential for EGT production in mycobacteria and for long-term infection of murine macrophages by M. tb. In this work, library screening and structure-guided strategies identified multiple classes of M. tb EgtD inhibitors that bind in various regions of the enzyme active site. X-ray crystal structures of EgtD-inhibitor complexes confirm that L-Histidine analogs bind solely to the L-Histidine binding site while drug-like inhibitors, such as TGX-221, and S-Glycyl-H-1152 span both the L-Histidine and AdoMet binding sites. These enzyme-inhibitor complexes provide detailed structural information of compound scaffolds useful for developing more potent inhibitors that could shorten Tuberculosis treatment regimens by weakening important bacterial defenses.


PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0244166
Author(s):  
Marco R. Rink ◽  
Marisa A. P. Baptista ◽  
Felix J. Flomm ◽  
Thomas Hennig ◽  
Adam W. Whisnant ◽  
...  

Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.


2021 ◽  
Vol 22 (15) ◽  
pp. 8048
Author(s):  
Raju Dash ◽  
Sarmistha Mitra ◽  
Yeasmin Akter Munni ◽  
Ho Jin Choi ◽  
Md. Chayan Ali ◽  
...  

An enzyme of the mammalian amino-sugar metabolism pathway, N-acetylglucosamine kinase (NAGK), that synthesizes N-acetylglucosamine (GlcNAc)-6-phosphate, is reported to promote dynein functions during mitosis, axonal and dendritic growth, cell migration, and selective autophagy, which all are unrelated to its enzyme activity. As non-enzymatic structural functions can be altered by genetic variation, we made an effort in this study aimed at deciphering the pathological effect of nonsynonymous single-nucleotide polymorphisms (nsSNPs) in NAGK gene. An integrated computational approach, including molecular dynamics (MD) simulation and protein–protein docking simulation, was used to identify the damaging nsSNPs and their detailed structural and functional consequences. The analysis revealed the four most damaging variants (G11R, G32R, G120E, and A156D), which are highly conserved and functional, positioned in both small (G11R and G32R) and large (G120E and A156D) domains of NAGK. G11R is located in the ATP binding region, while variants present in the large domain (G120E and A156D) were found to induce substantial alterations in the structural organizations of both domains, including the ATP and substrate binding sites. Furthermore, all variants were found to reduce binding energy between NAGK and dynein subunit DYNLRB1, as revealed by protein–protein docking and MM-GBSA binding energy calculation supporting their deleteriousness on non-canonical function. We hope these findings will direct future studies to gain more insight into the role of these variants in the loss of NAGK function and their role in neurodevelopmental disorders.


Author(s):  
Amrita Rai ◽  
Johann P. Klare ◽  
Patrick Y.A. Reinke ◽  
Felix Englmaier ◽  
Jörg Fohrer ◽  
...  

A novel cytoplasmic dye decolorizing peroxidase from Dictyostelium discoideum was investigated for its activity towards lignin oxidation. In contrast to related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer and oxidizes anthraquinone dyes, lignin model compounds and general peroxidase substrates like ABTS efficiently. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the distal and a water molecule at the axial face. Asp149 is in an optimal position to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long range electron transfer pathways associated with a hydrogen bonding network that connects the substrate binding sites with the heme moiety are described.


2021 ◽  
Author(s):  
orkun s soyer ◽  
Elisenda Feliu ◽  
Clarmyra Hayes

Here, we focus on a common class of enzymes that have multiple substrate-binding sites (multi-site enzymes), and analyse their capacity to generate bistable dynamics in the reaction systems that they are embedded in. Using mathematical techniques, we show that the inherent binding and catalysis reactions arising from multiple substrate-enzyme complexes creates a potential for bistable dynamics in a reaction system. We construct a generic model of an enzyme with n substrate binding sites and derive an analytical solution for the steady state concentration of all enzyme-substrate complexes. By studying these expressions, we obtain a mechanistic understanding for bistability and derive parameter combinations that guarantee bistability and show how changing specific enzyme kinetic parameters and enzyme levels can lead to bistability in reaction systems involving mjulti-site enzymes. Thus, the presented findings provide a biochemical and mathematical basis for predicting and engineering bistability in multi-site enzymes.


2021 ◽  
Vol 35 (S1) ◽  
Author(s):  
Hristina Zhekova ◽  
Alexander Pushkin ◽  
Gülru Kayık ◽  
Liyo Kao ◽  
Rustam Azimov ◽  
...  

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