Identification of a new nucleotide binding site by structural alignment and site directed mutagenesis

2018 ◽  
Vol 86 (11) ◽  
pp. 1140-1146
Author(s):  
Xiaoying Zhang ◽  
Qingzhan Yang ◽  
Qingya Shen ◽  
Jimin Zheng ◽  
Zongchao Jia
Keyword(s):  
Binding Site ◽  
Structural Alignment ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals The Nucleotide-binding Site of Human Sphingosine Kinase 1

2002 ◽  
Vol 277 (51) ◽  
pp. 49545-49553 ◽  
Author(s):  
Stuart M. Pitson ◽  
Paul A. B. Moretti ◽  
Julia R. Zebol ◽  
Reza Zareie ◽  
Claudia K. Derian ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Sphingosine Kinase ◽  
Structural Similarity ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Site Directed Mutagenesis ◽  
Sequence Motifs ◽  
Sphingosine Kinase 1 ◽  
Nucleotide Binding Sites

Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses including mitogenesis, anti-apoptosis, and expression of inflammatory molecules. Despite the importance of sphingosine kinase, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase does not contain recognizable catalytic or substrate-binding sites, based on sequence motifs found in other kinases. Here we have elucidated the nucleotide-binding site of human sphingosine kinase 1 (hSK1) through a combination of site-directed mutagenesis and affinity labeling with the ATP analogue, FSBA. We have shown that Gly82of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an ∼45-fold higherKm(ATP). We have also shown that Lys103is important in catalysis since an alanine substitution of this residue ablates catalytic activity. Furthermore, we have shown that this residue is covalently modified by FSBA. Our data, combined with amino acid sequence comparison, suggest a motif of SGDGX17–21K is involved in nucleotide binding in the sphingosine kinases. This motif differs in primary sequence from all previously identified nucleotide-binding sites. It does, however, share some sequence and likely structural similarity with the highly conserved glycine-rich loop, which is known to be involved in anchoring and positioning the nucleotide in the catalytic site of many protein kinases.

Characterization of Nucleotide Binding ­Site-Encoding Genes in Sweetpotato, Ipomoea batatas(L.) Lam., and Their Response to Biotic and Abiotic Stresses

2021 ◽  
pp. 1-15
Author(s):  
Zengzhi Si ◽  
Yake Qiao ◽  
Kai Zhang ◽  
Zhixin Ji ◽  
Jinling Han
Keyword(s):  
Binding Site ◽  
Abiotic Stresses ◽  
Ipomoea Batatas ◽  
Expression Profiles ◽  
Nucleotide Binding ◽  
Regulatory Elements ◽  
Nucleotide Binding Site ◽  
Biotic And Abiotic Stresses ◽  
Encoding Genes

Sweetpotato, <i>Ipomoea batatas</i> (L.) Lam., is an important and widely grown crop, yet its production is affected severely by biotic and abiotic stresses. The nucleotide binding site (NBS)-encoding genes have been shown to improve stress tolerance in several plant species. However, the characterization of NBS-encoding genes in sweetpotato is not well-documented to date. In this study, a comprehensive analysis of NBS-encoding genes has been conducted on this species by using bioinformatics and molecular biology methods. A total of 315 NBS-encoding genes were identified, and 260 of them contained all essential conserved domains while 55 genes were truncated. Based on domain architectures, the 260 NBS-encoding genes were grouped into 6 distinct categories. Phylogenetic analysis grouped these genes into 3 classes: TIR, CC (I), and CC (II). Chromosome location analysis revealed that the distribution of NBS-encoding genes in chromosomes was uneven, with a number ranging from 1 to 34. Multiple stress-related regulatory elements were detected in the promoters, and the NBS-encoding genes’ expression profiles under biotic and abiotic stresses were obtained. According to the bioinformatics analysis, 9 genes were selected for RT-qPCR analysis. The results revealed that <i>IbNBS75</i>, <i>IbNBS219</i>, and <i>IbNBS256</i> respond to stem nematode infection; <i>Ib­NBS240</i>, <i>IbNBS90</i>, and <i>IbNBS80</i> respond to cold stress, while <i>IbNBS208</i>, <i>IbNBS71</i>, and <i>IbNBS159</i> respond to 30% PEG treatment. We hope these results will provide new insights into the evolution of NBS-encoding genes in the sweetpotato genome and contribute to the molecular breeding of sweetpotato in the future.

Sign in / Sign up

Export Citation Format

Share Document