scholarly journals Identification of Key Amino Acid Residues Determining Product Specificity of 2,3-Oxidosqualene Cyclase in Siraitia grosvenorii

Catalysts ◽  
2018 ◽  
Vol 8 (12) ◽  
pp. 577 ◽  
Author(s):  
Jing Qiao ◽  
Jiushi Liu ◽  
Jingjing Liao ◽  
Zuliang Luo ◽  
Xiaojun Ma ◽  
...  
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Bioactive Molecules ◽  
Amino Acid Residues ◽  
Product Specificity ◽  
Oxidosqualene Cyclase ◽  
Siraitia Grosvenorii ◽  
Initial Substrate ◽  
New Strategy ◽  
Key Residues

Sterols and triterpenes are structurally diverse bioactive molecules generated through cyclization of linear 2,3-oxidosqualene. Based on carbocationic intermediates generated during the initial substrate preorganization step, oxidosqualene cyclases (OSCs) are roughly segregated into a dammarenyl cation group that predominantly catalyzes triterpenoid precursor products and a protosteryl cation group which mostly generates sterol precursor products. The mechanism of conversion between two scaffolds is not well understood. Previously, we have characterized a promiscuous OSC from Siraitia grosvenorii (SgCS) that synthesizes a novel cucurbitane-type triterpene cucurbitadienol as its main product. By integration of homology modeling, molecular docking and site-directed mutagenesis, we discover that five key amino acid residues (Asp486, Cys487, Cys565, Tyr535, and His260) may be responsible for interconversions between chair–boat–chair and chair–chair–chair conformations. The discovery of euphol, dihydrolanosterol, dihydroxyeuphol and tirucallenol unlocks a new path to triterpene diversity in nature. Our findings also reveal mechanistic insights into the cyclization of oxidosqualene into cucurbitane-type and lanostane-type skeletons, and provide a new strategy to identify key residues determining OSC specificity.

scholarly journals Protein engineering of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4

1993 ◽  
Vol 292 (1) ◽  
pp. 69-74 ◽  
Author(s):  
W Asmara ◽  
U Murdiyatmo ◽  
A J Baines ◽  
A T Bull ◽  
D J Hardman
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Protonated Form ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Pseudomonas Cepacia ◽  
Amino Acid Residues ◽  
Substrate Complex ◽  
Key Residues

The chemical modification of L-2-haloacid halidohydrolase IVa (Hdl IVa), originally identified in Pseudomonas cepacia MBA4, produced as a recombinant protein in Escherichia coli DH5 alpha, led to the identification of histidine and arginine as amino acid residues likely to play a part in the catalytic mechanism of the enzyme. These results, together with DNA sequence and analyses [Murdiyatmo, Asmara, Baines, Bull and Hardman (1992) Biochem. J. 284, 87-93] provided the basis for the rational design of a series of random- and site-directed-mutagenesis experiments of the Hdl IVa structural gene (hdl IVa). Subsequent apparent kinetic analyses of purified mutant enzymes identified His-20 and Arg-42 as the key residues in the activity of this halidohydrolase. It is also proposed that Asp-18 is implicated in the functioning of the enzyme, possibly by positioning the correct tautomer of His-20 for catalysis in the enzyme-substrate complex and stabilizing the protonated form of His-20 in the transition-state complex. Comparison of conserved amino acid sequences between the Hdl IVa and other halidohydrolases suggests that L-2-haloacid halidohydrolases contain conserved amino acid sequences that are not found in halidohydrolases active towards both D- and L-2-monochloropropionate.

scholarly journals Rapid resensitization of ASIC2a is conferred by three amino acid residues in the N terminus

2019 ◽  
Vol 151 (7) ◽  
pp. 944-953
Author(s):  
Jae Seung Lee ◽  
Hae-Jin Kweon ◽  
Hyosang Lee ◽  
Byung-Chang Suh
Keyword(s):  
Amino Acid ◽  
De Novo ◽  
Amino Acid Level ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Transmembrane Region ◽  
Acid Sensing Ion Channels ◽  
Electrophysiological Responses ◽  
N Terminus ◽  
Key Residues

Acid-sensing ion channels (ASICs), sensory molecules that continuously monitor the concentration of extracellular protons and initiate diverse intracellular responses through an influx of cations, are assembled from six subtypes that can differentially combine to form various trimeric channel complexes and elicit unique electrophysiological responses. For instance, homomeric ASIC1a channels have been shown to exhibit prolonged desensitization, and acid-evoked currents become smaller when the channels are repeatedly activated by extracellular protons, whereas homomeric or heteromeric ASIC2a channels continue to respond to repetitive acidic stimuli without exhibiting such desensitization. Although previous studies have provided evidence that both the desensitization of ASIC1a and rapid resensitization of ASIC2a commonly require domains that include the N terminus and the first transmembrane region of these channels, the biophysical basis of channel gating at the amino acid level has not been clearly determined. Here, we confirm that domain-swapping mutations replacing the N terminus of ASIC2a with that of ASIC2b result in de novo prolonged desensitization in homomeric channels following activation by extracellular protons. Such desensitization of chimeric ASIC2a mutants is due neither to internalization nor to degradation of the channel proteins. We use site-directed mutagenesis to narrow down the relevant portion of the N terminus of ASIC2a, identifying three amino acid residues within the N terminus (T25, T39, and I40) whose mutation is sufficient to phenocopy the desensitization exhibited by the chimeric mutants. A similar desensitization is observed in heteromeric ASICs containing the mutant subunit. These results suggest that T25, T39, and I40 of ASIC2a are key residues determining the rapid resensitization of homomeric and heteromeric ASIC2a channels upon proton activation.

scholarly journals Identification of key amino acid residues determining product specificity of 2,3-oxidosqualene cyclase in Oryza species

2018 ◽  
Vol 218 (3) ◽  
pp. 1076-1088 ◽  
Author(s):  
Zheyong Xue ◽  
Zhengwei Tan ◽  
Ancheng Huang ◽  
Yuan Zhou ◽  
Juncong Sun ◽  
...  
Keyword(s):  
Amino Acid ◽  
Oryza Species ◽  
Amino Acid Residues ◽  
Product Specificity ◽  
Oxidosqualene Cyclase

scholarly journals Defining key residues of the Swi1 prion domain in prion formation and maintenance

2021 ◽  
Author(s):  
Dustin K. Goncharoff ◽  
Raudel Cabral ◽  
Sarah V. Applebey ◽  
Manasa Pagadala ◽  
Zhiqiang Du ◽  
...  
Keyword(s):  
Amino Acid ◽  
De Novo ◽  
Neurological Diseases ◽  
Low Complexity ◽  
Small Region ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Cellular Functions ◽  
Alternative Protein ◽  
Key Residues

Prions are self-perpetuating, alternative protein conformations associated with neurological diseases and normal cellular functions. Saccharomyces cerevisiae contains many endogenous prions – providing a powerful system to study prionization. Previously, we demonstrated that Swi1, a component of the SWI/SNF chromatin-remodeling complex, can form the prion [SWI+]. A small region, Swi11-38, with a unique amino-acid composition of low complexity, acts as a prion domain and supports [SWI+] propagation. Here, we further examine Swi11-38 through site-directed mutagenesis. We found that mutations of the two phenylalanine residues or threonine tract inhibit Swi11-38 aggregation. In addition, mutating both phenylalanines can abolish de novo prion formation by Swi11-38 whereas mutating only one phenylalanine does not. Replacement of half or the entire eight-threonine tract with alanines has the same effect, possibly disrupting a core region of Swi11-38 aggregates. We also show that Swi11-38 and its prion-fold-maintaining mutants form high-molecular-weight, SDS-resistant aggregates whereas the double phenylalanine mutants eliminate these protein species. These results indicate the necessity of the large hydrophobic residues and threonine tract in Swi11-38 in prionogenesis – possibly acting as important aggregatable regions. Our findings thus highlight the importance of specific amino-acid residues in the Swi1 prion domain in prion formation and maintenance.

scholarly journals Mutation of Key Residues in β-Glycosidase LXYL-P1-2 for Improved Activity

Catalysts ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1042
Author(s):  
Jing-Jing Chen ◽  
Xiao Liang ◽  
Tian-Jiao Chen ◽  
Jin-Ling Yang ◽  
Ping Zhu
Keyword(s):  
Amino Acid ◽  
Lentinula Edodes ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Recent Success ◽  
Key Residues ◽  
Positive Effect ◽  
Candidate Strain

The β-glycosidase LXYL-P1-2 identified from Lentinula edodes can be used to hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) into 10-deacetyltaxol (DT) for the semi-synthesis of Taxol. Recent success in obtaining the high-resolution X-ray crystal of LXYL-P1-2 and resolving its three-dimensional structure has enabled us to perform molecular docking of LXYL-P1-2 with substrate XDT and investigate the roles of the three noncatalytic amino acid residues located around the active cavity in LXYL-P1-2. Site-directed mutagenesis results demonstrated that Tyr268 and Ser466 were essential for maintaining the β-glycosidase activity, and the L220G mutation exhibited a positive effect on increasing activity by enlarging the channel that facilitates the entrance of the substrate XDT into the active cavity. Moreover, introducing L220G mutation into the other LXYL-P1-2 mutant further increased the enzyme activity, and the β-d-xylosidase activity of the mutant EP2-L220G was nearly two times higher than that of LXYL-P1-2. Thus, the recombinant yeast GS115-EP2-L220G can be used for efficiently biocatalyzing XDT to DT for the semi-synthesis of Taxol. Our study provides not only the prospective candidate strain for industrial production, but also a theoretical basis for exploring the key amino acid residues in LXYL-P1-2.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

scholarly journals Identification and characterization of amphibian SLC26A5 using RNA-Seq

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhongying Wang ◽  
Qixuan Wang ◽  
Hao Wu ◽  
Zhiwu Huang
Keyword(s):  
Amino Acid ◽  
Inner Ear ◽  
Hair Cells ◽  
Rana Catesbeiana ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Rna Seq ◽  
American Bullfrog ◽  
Frog Species

Abstract Background Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified. Results Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana’s inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog’s prestin was functionally different from Rana. Conclusions We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin.

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