scholarly journals Two Highly Similar Chitinases from Marine Vibrio Species have Different Enzymatic Properties

Marine Drugs ◽  
2020 ◽  
Vol 18 (3) ◽  
pp. 139
Author(s):  
Xinxin He ◽  
Min Yu ◽  
Yanhong Wu ◽  
Lingman Ran ◽  
Weizhi Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Extracellular Enzymes ◽  
Vibrio Harveyi ◽  
Amino Acid Sequences ◽  
Enzymatic Properties ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Marine Vibrio ◽  
Key Sites

Chitinase, as one of the most important extracellular enzymes in the marine environment, has great ecological and applied values. In this study, two chitinases (Chi1557 and Chi4668) with 97.33% amino acid sequences identity were individually found in Vibrio rotiferianus and Vibrio harveyi. They both were encoding by 561 amino acids, but differed in 15 amino acids and showed different enzymatic properties. The optimal temperature and pH ranges were 45–50 °C and pH 5.0–7.0 for Chi1557, while ~50 °C and pH 3.0–6.0 for Chi4668. K+, Mg2+, and EDTA increased the enzymatic activity of Chi4668 significantly, yet these factors were inhibitory to Chi1557. Moreover, Chi1557 degraded colloidal chitin to produce (GlcNAc)2 and minor GlcNAc, whereas Chi4668 produce (GlcNAc)2 with minor (GlcNAc)3 and (GlcNAc)4. The Kcat/Km of Chi4668 was ~4.7 times higher than that of Chi1557, indicating that Chi4668 had stronger catalytic activity than Chi1557. Furthermore, site-directed mutagenesis was performed on Chi1557 focusing on seven conserved amino acid residues of family GH18 chitinases. Chi1557 was almost completely inactive after Glu154, Gln219, Tyr221, or Trp312 was individually mutated, retained ~50% activity after Tyr37 was mutated, and increased two times activity after Asp152 was mutated, indicating that these six amino acids were key sites for Chi1557.

Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

2000 ◽  
Vol 349 (1) ◽  
pp. 281-287 ◽  
Author(s):  
Patricia E. M. MARTIN ◽  
James STEGGLES ◽  
Claire WILSON ◽  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Lucifer Yellow ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

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 iSulfoTyr-PseAAC: Identify Tyrosine Sulfation Sites by Incorporating Statistical Moments via Chou’s 5-steps Rule and Pseudo Components

2019 ◽  
Vol 20 (4) ◽  
pp. 306-320 ◽  
Author(s):  
Omar Barukab ◽  
Yaser Daanial Khan ◽  
Sher Afzal Khan ◽  
Kuo-Chen Chou
Keyword(s):  
Amino Acid ◽  
Computational Model ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Statistical Moments ◽  
Amino Acid Residues ◽  
Tyrosine Sulfation ◽  
Protein Amino Acid ◽  
Jackknife Test ◽  
Self Consistency

Background: The amino acid residues, in protein, undergo post-translation modification (PTM) during protein synthesis, a process of chemical and physical change in an amino acid that in turn alters behavioral properties of proteins. Tyrosine sulfation is a ubiquitous posttranslational modification which is known to be associated with regulation of various biological functions and pathological processes. Thus its identification is necessary to understand its mechanism. Experimental determination through site-directed mutagenesis and high throughput mass spectrometry is a costly and time taking process, thus, the reliable computational model is required for identification of sulfotyrosine sites. Methodology: In this paper, we present a computational model for the prediction of the sulfotyrosine sites named iSulfoTyr-PseAAC in which feature vectors are constructed using statistical moments of protein amino acid sequences and various position/composition relative features. These features are incorporated into PseAAC. The model is validated by jackknife, cross-validation, self-consistency and independent testing. Results: Accuracy determined through validation was 93.93% for jackknife test, 95.16% for crossvalidation, 94.3% for self-consistency and 94.3% for independent testing. Conclusion: The proposed model has better performance as compared to the existing predictors, however, the accuracy can be improved further, in future, due to increasing number of sulfotyrosine sites in proteins.

scholarly journals Conserved Repeat Motifs and Glucan Binding by Glucansucrases of Oral Streptococci and Leuconostoc mesenteroides

2004 ◽  
Vol 186 (24) ◽  
pp. 8301-8308 ◽  
Author(s):  
Deepan S. H. Shah ◽  
Gilles Joucla ◽  
Magali Remaud-Simeon ◽  
Roy R. B. Russell
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Proteins ◽  
Leuconostoc Mesenteroides ◽  
Aromatic Amino Acids ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Oral Streptococci ◽  
Basic Residue ◽  
Essential Contribution

ABSTRACT Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly α-1,3 or α-1,6 links, as well as alternating α-1,3 and α-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.

scholarly journals Amino Acid Residues Essential for Function of the MexF Efflux Pump Protein of Pseudomonas aeruginosa

2002 ◽  
Vol 46 (7) ◽  
pp. 2169-2173 ◽  
Author(s):  
Julio Ramos Aires ◽  
Jean-Claude Pechère ◽  
Christian Van Delden ◽  
Thilo Köhler
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Efflux Pump ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Proper Function ◽  
Amino Acid Residues ◽  
Pump System ◽  
Transmembrane Segments ◽  
Partial Activity

ABSTRACT At least four broad-spectrum efflux pumps (Mex) are involved in elevated intrinsic antibiotic resistance as well as in acquired multidrug resistance in Pseudomonas aeruginosa. Substrate specificity of the Mex pumps has been shown to be determined by the cytoplasmic membrane component (MexB, MexD, MexF, and MexY) of the tripartite efflux pump system. Alignment of their amino acid sequences with those of the homologous AcrB and AcrD pump proteins of Escherichia coli showed conservation of five charged amino acid residues located in or next to transmembrane segments (TMS). These residues were mutated in the MexF gene by site-directed mutagenesis and replaced by residues of opposite or neutral charge. MexF proteins containing combined D410A and A411G substitutions located in TMS4 were completely inactive. Similarly, the substitutions E417K (next to TMS4) and K951E (TMS10) also caused loss of activity towards all tested antibiotics. The substitution E349K in TMS2 resulted in a MexF mutant protein which was unable to transport trimethoprim and quinolones but retained partial activity for the transport of chloramphenicol. All mutated MexF proteins were expressed at comparable levels when tested by Western blot analysis. It is concluded that charged residues located in or close to TMS are essential for proper function of MexF.

scholarly journals A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

2013 ◽  
Vol 289 (3) ◽  
pp. 1377-1387 ◽  
Author(s):  
Jagdeep Kaur ◽  
Elena Olkhova ◽  
Viveka Nand Malviya ◽  
Ernst Grell ◽  
Hartmut Michel
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ligand Binding ◽  
Isothermal Titration Calorimetry ◽  
Functional Characterization ◽  
Homology Model ◽  
Docking Studies ◽  
Site Directed Mutagenesis ◽  
Titration Calorimetry ◽  
Amino Acid Residues

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.

scholarly journals Molecular Cloning and Bioinformatics Analysis of DQA Gene from Mink (Neovison vison)

2019 ◽  
Vol 20 (5) ◽  
pp. 1037
Author(s):  
Zhaobin Fan ◽  
Houfeng Zhang ◽  
Min Rong ◽  
Dongmei Meng ◽  
Zhenxing Yu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mechanisms ◽  
Signal Sequence ◽  
Amino Acid Sequences ◽  
Neovison Vison ◽  
Random Coil ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Full Length Sequence

In the present study, we cloned, sequenced, and explored the structural and functional characteristics of the major histocompatibility complex (MHC)-DQA gene from mink (Neovison vison) for the first time. The full-length sequence of DQA gene was 1147-bp-long, contained a coding region of 768-bp, which was predicted to encoding 255 amino acid residues. The comparison between DQA from mink (Neovison vison) and other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of the mink DQA gene exhibited high similarity with the ferret (Mustela pulourius furo). Phylogenetic analysis revealed that mink (Neovison vison) DQA is grouped with that of ferret (Mustela pulourius furo). The cloned sequence contained a 23-amino acid NH2-terminal signal sequence with the signal peptide cutting site located in amino acids 23–24, and had three Asn-Xaa-Ser/Thr sequons. Three cysteine residues were also identified (Cys-85, Cys-121, and Cys-138). The 218 to 240 amino acids were predicted to be the transmembrane domains. The prediction of the secondary structure revealed three α-helixes and fourteen β-sheets in Neovison vison DQA protein, while random coil was a major pattern. In this study, the whole CDS sequence of Neovison vison DQA gene was successfully cloned, which was valuable for exploring the function and antiviral molecular mechanisms underlying the molecule. The findings of the present study have laid the foundation for the disease resistance and breeding of mink.

scholarly journals Enzymatic Properties of Dipeptidyl Aminopeptidase IV Produced by the Periodontal Pathogen Porphyromonas gingivalis and Its Participation in Virulence

2000 ◽  
Vol 68 (2) ◽  
pp. 716-724 ◽  
Author(s):  
Yumi Kumagai ◽  
Kiyoshi Konishi ◽  
Tomoharu Gomi ◽  
Hisao Yagishita ◽  
Ayako Yajima ◽  
...  
Keyword(s):  
Amino Acid ◽  
Porphyromonas Gingivalis ◽  
Catalytic Domain ◽  
Animal Experiments ◽  
Catalytic Triad ◽  
Amino Acid Sequences ◽  
Enzymatic Properties ◽  
Site Directed Mutagenesis ◽  
Periodontal Pathogen ◽  
Dipeptidyl Aminopeptidase

ABSTRACT Porphyromonas gingivalis is a major pathogen associated with adult periodontitis. We cloned and sequenced the gene (dpp) coding for dipeptidyl aminopeptidase IV (DPPIV) fromP. gingivalis W83, based on the amino acid sequences of peptide fragments derived from purified DPPIV. An Escherichia coli strain overproducing P. gingivalis DPPIV was constructed. The enzymatic properties of recombinant DPPIV purified from the overproducer were similar to those of DPPIV isolated fromP. gingivalis. The three amino acid residues Ser, Asp, and His, which are thought to form a catalytic triad in the C-terminal catalytic domain of eukaryotic DPPIV, are conserved in P. gingivalis DPPIV. When each of the corresponding residues of the enzyme was substituted with Ala by site-directed mutagenesis, DPPIV activity significantly decreased, suggesting that these three residues of P. gingivalis DPPIV are involved in the catalytic reaction. DPPIV-deficient mutants of P. gingivalis were constructed and subjected to animal experiments. Mice injected with the wild-type strain developed abscesses to a greater extent and died more frequently than those challenged with mutant strains. Mice injected with the mutants exhibited faster recovery from the infection, as assessed by weight gain and the rate of lesion healing. This decreased virulence of mutants compared with the parent strain suggests that DPPIV is a potential virulence factor of P. gingivalis and may play important roles in the pathogenesis of adult periodontitis induced by the organism.

scholarly journals Introduction of H-2Dd determinants into the H-2Ld antigen by site-directed mutagenesis.

1987 ◽  
Vol 166 (3) ◽  
pp. 744-760 ◽  
Author(s):  
D Koeller ◽  
R Lieberman ◽  
J Miyazaki ◽  
E Appella ◽  
K Ozato ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mhc Class I ◽  
Amino Acid Position ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Antigenic Sites ◽  
Vesicular Stomatitis ◽  
Mhc Class I Antigen

We used site-directed mutagenesis to localize serologically defined (s) and CTL (c)-defined alloantigenic determinants to discrete amino acid sequences of a murine MHC class I antigen. Based on the prediction that amino acid position 63-73 of the H-2Dd antigen forms s-allodeterminants, the H-2Ld gene was mutated in a sequential fashion to replace codons for amino acid positions 63, 65, 66, 70, and 73 with those of the H-2Dd amino acids. Epitopes of the mutant antigens expressed in L-cells were examined by the binding of a series of mAbs specific for the H-2Dd antigen. The mutant antigen M66 had substitutions at residues 63, 65, and 66, and resulted in the acquisition of a number of H-2Dd-specific s-epitopes. Mutant M70 had an additional substitution at residue 70, which led to the gain of multiple additional H-2Dd s-epitopes. Together, more than half of all the relevant H-2Dd s-epitopes were mapped into amino acid position 63-70 of the H-2Dd molecule, which was expressed in the mutant H-2Ld gene. The final mutation at residue 73 (M73) caused no new epitope gains, rather, a few Dd s-epitopes acquired by the preceding mutations were lost. All of the H-2Ld-specific s-determinants were retained in the mutant molecules, as were H-2Dd s-determinants specific for the alpha-2 or alpha-3 domains. Changes of these residues affected c-determinants defined by CTL. Anti-H-2Dd CTL cultures and an anti-H-2Dd CTL clone recognized the mutant H-2Ld molecules, M66 and M70. Some CTL clones generated against the Q10d molecule, which has an identical sequence to H-2Dd between residues 61 and 73, failed to recognize native H-2Dd or Ld but did crossreact with mutant Ld. While bulk-cultured anti-H-2Ld CTL cultures reacted strongly against M73, bulk-cultured H-2Ld restricted anti-vesicular stomatitis virus CTL did not. Finally, at the clonal level two of three anti-H-2Ld CTL clones lost reactivity with some or all of these mutant molecules. From these results we conclude that a stretch of amino acids from position 63 to 70 of the alpha-1 domain controls major s- and c-antigenic sites on the H-2Dd antigen and c-sites on H-2Ld antigen.

scholarly journals Combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) in the coat protein of apple chlorotic leaf spot virus are crucial for infectivity

2007 ◽  
Vol 88 (9) ◽  
pp. 2611-2618 ◽  
Author(s):  
Hajime Yaegashi ◽  
Masamichi Isogai ◽  
Hiroko Tajima ◽  
Teruo Sano ◽  
Nobuyuki Yoshikawa
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Coat Protein ◽  
Leaf Spot ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Cdna Clones ◽  
Spot Virus ◽  
Chlorotic Leaf

Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala40-Val59-Phe75-Ser130-Met184 or Ser40-Leu59-Tyr75-Thr130-Leu184) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala40 to Ser40 or Phe75 to Tyr75) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.

Sign in / Sign up

Export Citation Format

Share Document