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.

scholarly journals In Vivo and In Vitro Studies of Bacillus subtilis Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway

2002 ◽  
Vol 184 (14) ◽  
pp. 4018-4024 ◽  
Author(s):  
Ulf Olsson ◽  
Annika Billberg ◽  
Sara Sjövall ◽  
Salam Al-Karadaghi ◽  
Mats Hansson
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Three Dimensional Structure ◽  
Activity Measurements

ABSTRACT Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix α3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of B. subtilis and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in V max. The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.

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.

Characteristics of Two Forms of α-Amylases and Structural Implication

1999 ◽  
Vol 65 (10) ◽  
pp. 4652-4658 ◽  
Author(s):  
Kohji Ohdan ◽  
Takashi Kuriki ◽  
Hiroki Kaneko ◽  
Jiro Shimada ◽  
Toshikazu Takada ◽  
...  
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Amylase Gene ◽  
Raw Starch ◽  
Raw Starch Binding ◽  
Terminal Amino ◽  
Carboxyl Terminal

ABSTRACT Complete (Ba-L) and truncated (Ba-S) forms of α-amylases fromBacillus subtilis X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined. The amino acid sequence deduced from the nucleotide sequence of the α-amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region. The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same α-amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of B. subtilis X-23 cells. Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L. An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as α-amylase.

scholarly journals Efficacy of Dideoxynucleosides against Human Foamy Virus and Relationship to Its Reverse Transcriptase Amino Acid Sequence and Structure

2001 ◽  
Vol 75 (15) ◽  
pp. 7184-7187 ◽  
Author(s):  
Anne Yvon-Groussin ◽  
Pierre Mugnier ◽  
Philippe Bertin ◽  
Marc Grandadam ◽  
Henri Agut ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reverse Transcriptase ◽  
Foamy Virus ◽  
Three Dimensional ◽  
Retroviral Vectors ◽  
Dimensional Structure ◽  
Human Foamy Virus ◽  
Amino Acid Residues ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human foamy virus (HFV), a retrovirus of simian origin which occasionally infects humans, is the basis of retroviral vectors in development for gene therapy. Clinical considerations of how to treat patients developing an uncontrolled infection by either HFV or HFV-based vectors need to be raised. We determined the susceptibility of the HFV to dideoxynucleosides and found that only zidovudine was equally efficient against the replication of human immunodeficiency virus type 1 (HIV-1) and HFV. By contrast, zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), and didanosine (ddI) were 3-, 3-, 30-, and 46-fold less efficient against HFV than against HIV-1, respectively. Some amino acid residues known to be involved in HIV-1 resistance to ddC, 3TC, d4T, and ddI were found at homologous positions of HFV reverse transcriptase (RT). These critical amino acids are located at the same positions in the three-dimensional structure of HIV-1 and HFV RT, suggesting that both enzymes share common patterns of inhibition.

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 A Membrane-Bound Flavocytochromec-Sulfide Dehydrogenase from the Purple Phototrophic Sulfur Bacterium Ectothiorhodospira vacuolata

2000 ◽  
Vol 182 (11) ◽  
pp. 3097-3103 ◽  
Author(s):  
Vesna Kostanjevecki ◽  
Ann Brigé ◽  
Terrance E. Meyer ◽  
Michael A. Cusanovich ◽  
Yves Guisez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Signal Sequence ◽  
Sulfide Oxidation ◽  
Three Dimensional ◽  
Chromatium Vinosum ◽  
Dimensional Structure ◽  
Northern Hybridization ◽  
Amino Acid Residues ◽  
Membrane Bound

ABSTRACT The amino acid sequence of Ectothiorhodospira vacuolatacytochrome c-552, isolated from membranes withn-butanol, shows that it is a protein of 77 amino acid residues with a molecular mass of 9,041 Da. It is closely related to the cytochrome subunit of Chlorobium limicola f. sp.thiosulfatophilum flavocytochrome c-sulfide dehydrogenase (FCSD), having 49% identity. These data allowed isolation of a 5.5-kb subgenomic clone which contains the cytochrome gene and an adjacent flavoprotein gene as in other species which have an FCSD. The cytochrome subunit has a signal peptide with a normal cleavage site, but the flavoprotein subunit has a signal sequence which suggests that the mature protein has an N-terminal cysteine, characteristic of a diacyl glycerol-modified lipoprotein. The membrane localization of FCSD was confirmed by Western blotting with antibodies raised against Chromatium vinosum FCSD. When aligned according to the three-dimensional structure of ChromatiumFCSD, all but one of the side chains near the flavin are conserved. These include the Cys 42 flavin adenine dinucleotide binding site; the Cys 161-Cys 337 disulfide; Glu 167, which modulates the reactivity with sulfite; and aromatic residues which may function as charge transfer acceptors from the flavin-sulfite adduct (C. vinosumnumbering). The genetic context of FCSD is different from that in other species in that flanking genes are not conserved. The transcript is only large enough to encode the two FCSD subunits. Furthermore, Northern hybridization showed that the production of E. vacuolata FCSD mRNA is regulated by sulfide. All cultures that contained sulfide in the medium had elevated levels of FCSD RNA compared with cells grown on organics (acetate, malate, or succinate) or thiosulfate alone, consistent with the role of FCSD in sulfide oxidation.

scholarly journals Site-directed mutagenesis of the putative active site of human 17β-hydroxysteroid dehydrogenase type 1

1994 ◽  
Vol 304 (1) ◽  
pp. 289-293 ◽  
Author(s):  
T J Puranen ◽  
M H Poutanen ◽  
H E Peltoketo ◽  
P T Vihko ◽  
R K Vihko
Keyword(s):  
Amino Acid ◽  
Catalytic Properties ◽  
Catalytic Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme

Several amino acid residues (Cys54, Tyr155, His210, His213 and His221) at a putative catalytic site of human 17 beta-hydroxysteroid dehydrogenase type 1 were mutated to Ala. Replacement of His221 by Ala remarkably reduced the catalytic activity, which resulted from a change of both the Km and the Vmax. values of the enzyme. Compared with the wild-type enzyme, the catalytic efficiency of the His221-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His210 or His213, no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the Vmax. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys54 has been localized in the cofactor-binding region of the enzyme, a Cys54-->Ala mutation did not lead to changes in the enzymic activity. The most dramatic effects on the catalytic properties of the enzyme were achieved by mutating Tyr155, which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Tyr155-->Ala, His210-->Ala + His213-->Ala and His221-->Ala mutations were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lower catalytic efficiency of the mutant enzymes is due to an exchange of catalytically important amino acid residues and/or remarkable alterations in the three-dimensional structure of the enzyme. The recently detected polymorphisms (Ala237<-->Val and Ser312<-->Gly) were not found to affect either the catalytic or the immunological properties of the type 1 enzyme.

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.

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