Implications of Stisa2 catalytic residue restoration through site directed mutagenesis

2016 ◽  
Vol 42 (2) ◽  
Author(s):  
Hasnain Hussain ◽  
Nikson Fatt Ming Chong
Keyword(s):  
Catalytic Activity ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Qualitative And Quantitative Analysis ◽  
Conserved Residues ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
Catalytic Network ◽  
And Function

AbstractObjective:Restoration of catalytic activity of Isa2 fromMethods:The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed inResults:Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity.Conclusions:These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity.

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 Intramolecular Interactions between the Protease and Structural Domains Are Important for the Functions of Serine Protease Autotransporters

2010 ◽  
Vol 78 (8) ◽  
pp. 3335-3345 ◽  
Author(s):  
Casey Tsang ◽  
Huma Malik ◽  
Deana Nassman ◽  
Antony Huang ◽  
Fayha Tariq ◽  
...  
Keyword(s):  
Serine Protease ◽  
Signal Sequence ◽  
Catalytic Triad ◽  
Intramolecular Interactions ◽  
Site Directed Mutagenesis ◽  
Temperature Sensitive ◽  
Molecular Graphics ◽  
Passenger Domain ◽  
Conserved Residues ◽  
Functional Roles

ABSTRACT Autotransporter (AT) is a protein secretion pathway found in Gram-negative bacteria featuring a multidomain polypeptide with a signal sequence, a passenger domain, and a translocator domain. An AT subfamily named serine protease ATs of the family Enterobacteriaceae (SPATEs) is characterized by the presence of a conserved serine protease motif in the passenger domain which contributes to bacterial pathogenesis. The goal of the current study is to determine the importance of the passenger domain conserved residues in the SPATE proteolytic and adhesive functions using the temperature-sensitive hemagglutinin (Tsh) protein as our model. To begin, mutations of 21 fully conserved residues in the four passenger domain conserved motifs were constructed by PCR-based site-directed mutagenesis. Seventeen mutants exhibited a wild-type secretion level; among these mutants, eight displayed reduced proteolytic activities in Tsh-specific oligopeptide and mucin cleavage assays. These eight mutants also demonstrated lower affinities to extracellular matrix proteins, collagen IV, and fibronectin. These eight conserved residues were analyzed by molecular graphics modeling to demonstrate their intramolecular interactions with the catalytic triad and other key residues. Additional mutations were made to confirm the above interactions in order to demonstrate their significance to the SPATE functions. Altogether our data suggest that certain conserved residues in the SPATE passenger domain are important for both the proteolytic and adhesive activities of SPATE by maintaining the proper protein structure via intramolecular interactions between the protease and β-helical domains. Here, we provide new insight into the structure-function relationship of the SPATEs and the functional roles of their conserved residues.

scholarly journals Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors

1997 ◽  
Vol 323 (2) ◽  
pp. 415-419 ◽  
Author(s):  
Lakshmi KASTURI ◽  
Hegang CHEN ◽  
Susan H. SHAKIN-ESHLEMAN
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Free Translation ◽  
A Cell ◽  
And Function ◽  
A Site ◽  
The Impact

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa = Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa = Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

Activity modulation of the oligopeptidase B from Serratia proteamaculans by site-directed mutagenesis of amino acid residues surrounding catalytic triad histidine

Biochimie ◽  
2017 ◽  
Vol 139 ◽  
pp. 125-136 ◽  
Author(s):  
Anna G. Mikhailova ◽  
Tatiana V. Rakitina ◽  
Vladimir I. Timofeev ◽  
David M. Karlinsky ◽  
Dmitry A. Korzhenevskiy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Serratia Proteamaculans ◽  
Oligopeptidase B

scholarly journals Identification of Highly Conserved Residues Involved in Inhibition of HIV-1 RNase H Function by Diketo Acid Derivatives

2014 ◽  
Vol 58 (10) ◽  
pp. 6101-6110 ◽  
Author(s):  
Angela Corona ◽  
Francesco Saverio Di Leva ◽  
Sylvain Thierry ◽  
Luca Pescatori ◽  
Giuliana Cuzzucoli Crucitti ◽  
...  
Keyword(s):  
Rational Design ◽  
Acid Ethyl Ester ◽  
Rnase H ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Conserved Residues ◽  
Essential Function ◽  
Catalytic Motif ◽  
Hiv 1 ◽  
Micromolar Range

ABSTRACTHIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg2+-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors.

scholarly journals Identification of critical amino acid residues in the plague biofilm Hms proteins

Microbiology ◽  
2006 ◽  
Vol 152 (11) ◽  
pp. 3399-3410 ◽  
Author(s):  
Stanislav Forman ◽  
Alexander G. Bobrov ◽  
Olga Kirillina ◽  
Susannah K. Craig ◽  
Jennifer Abney ◽  
...  
Keyword(s):  
Protein Function ◽  
Biofilm Formation ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
High Similarity ◽  
Conserved Residues ◽  
Periplasmic Domain ◽  
Effective Transmission

Yersinia pestis biofilm formation causes massive adsorption of haemin or Congo red in vitro as well as colonization and eventual blockage of the flea proventriculus in vivo. This blockage allows effective transmission of plague from some fleas, like the oriental rat flea, to mammals. Four Hms proteins, HmsH, HmsF, HmsR and HmsS, are essential for biofilm formation, with HmsT and HmsP acting as positive and negative regulators, respectively. HmsH has a β-barrel structure with a large periplasmic domain while HmsF possesses polysaccharide deacetylase and COG1649 domains. HmsR is a putative glycosyltransferase while HmsS has no recognized domains. In this study, specific amino acids within conserved domains or within regions of high similarity in HmsH, HmsF, HmsR and HmsS proteins were selected for site-directed mutagenesis. Some but not all of the substitutions in HmsS and within the periplasmic domain of HmsH were critical for protein function. Substitutions within the glycosyltransferase domain of HmsR and the deacetylase domain of HmsF abolished biofilm formation in Y. pestis. Surprisingly, substitution of highly conserved residues within COG1649 did not affect HmsF function.

scholarly journals Cloning, Expression, Characterization, and Antioxidant Protection of Glutaredoxin3 From Psychrophilic Bacterium Psychrobacter sp. ANT206

2021 ◽  
Vol 12 ◽  
Author(s):  
Yatong Wang ◽  
Yanhua Hou ◽  
Quanfu Wang
Keyword(s):  
Catalytic Activity ◽  
Point Mutations ◽  
Cd Spectroscopy ◽  
Biochemical Properties ◽  
Site Directed Mutagenesis ◽  
Psychrophilic Bacterium ◽  
Amino Acid Residues ◽  
Salt Bridges ◽  
Active Site Motif ◽  
Expression Characterization

Glutaredoxins (Grxs) are proteins that catalyze the glutathione (GSH)-dependent reduction of protein disulfides. In this study, a Grx-related gene (264 bp), encoding a Ps-Grx3, was cloned from Psychrobacter sp. ANT206. Sequence analysis indicated the presence of the active site motif CPYC in this protein. Homology modeling showed that Ps-Grx3 had fewer hydrogen bonds and salt bridges, as well as a lower Arg/(Arg + Lys) ratio than its mesophilic homologs, indicative of an improved catalytic ability at low temperatures. Site-directed mutagenesis demonstrated that the Cys13, Pro14, and Cys16 sites were essential for the catalytic activity of Ps-Grx3, while circular dichroism (CD) spectroscopy confirmed that point mutations in these amino acid residues led to the loss or reduction of enzyme activity. Furthermore, analysis of the biochemical properties of Ps-Grx3 showed that the optimum temperature of this enzyme was 25 °C. Importantly, Ps-Grx3 was more sensitive to tBHP and CHP than to H2O2, and retained approximately 40% activity even when the H2O2 concentration was increased to 1 mm Regarding substrate specificity, Ps-Grx3 had a higher affinity for HED, L-cystine, and DHA than for S-sulfocysteine and BSA. We also investigated the DNA-protective ability of Ps-Grx3 using the pUC19 plasmid, and found that Ps-Grx3 could protect supercoiled DNA from oxidation-induced damage at 15°C for 1.5 h. This study provides new insights into the structure and catalytic activity of a cold-adapted Grx3.

scholarly journals Mechanism of the CBM35 domain in assisting catalysis by Ape1, a Campylobacter jejuni O-acetyl esterase

2021 ◽  
Author(s):  
Chang Sheng-Huei Lin ◽  
Ian Y. Yen ◽  
Anson C. K. Chan ◽  
Michael E. P. Murphy
Keyword(s):  
Campylobacter Jejuni ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Binding Mode ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Acetyl Esterase ◽  
Lytic Transglycosylases ◽  
Active Site Cleft ◽  
And Function

AbstractPeptidoglycan (PG) is O-acetylated by bacteria to resist killing by host lysozyme. During PG turnover, however, deacetylation is a prerequisite for glycan strand hydrolysis by lytic transglycosylases. Ape1, a de-O-acetylase from Campylobacter jejuni, is a bi-modular protein composed of an SGNH hydrolase domain and a CBM35 domain. The conserved Asp-His-Ser catalytic triad in the SGNH hydrolase domain confers enzymatic activity. The PG binding mode and function of the CBM35 domain in de-O-acetylation remained unclear. In this paper, we present a 1.8 Å resolution crystal structure of a complex between acetate and Ape1. An active site cleft is formed at the interface of the two domains and two large loops from the CBM35 domain form part of the active site. Site-directed mutagenesis of residues in these loops coupled with activity assays using p-nitrophenol acetate indicate the CBM35 loops are required for full catalytic efficiency. Molecular docking of a model O-acetylated hexasaccharide PG substrate to Ape1 using HADDOCK suggests the interaction is formed by the active cleft and the saccharide motif of PG. Together, we propose that the active cleft of Ape1 diverges from other SGNH hydrolase members by using the CBM35 loops to assist catalysis. The concave Ape1 active cleft may accommodate the long glycan strands for selecting PG substrates to regulate subsequent biological events.

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