scholarly journals Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A

2003 ◽  
Vol 370 (1) ◽  
pp. 331-337 ◽  
Author(s):  
Paloma SÁNCHEZ-TORRES ◽  
Jaap VISSER ◽  
Jacques A.E. BENEN
Keyword(s):  
Aspergillus Niger ◽  
Conformational Change ◽  
Three Dimensional ◽  
Pectin Lyase ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Fluorescence Studies ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Positively Charged

Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed β-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH. On the basis of the three-dimensional structures of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677—689] and the modelled enzyme—substrate complex of PL1B [Herron, Benen, Scavetta, Visser and Jurnak (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 8762—8769], Asp154, Arg176, Arg236 and Lys239 were mutagenized. Substituting Arg236 with alanine or lysine rendered the enzyme completely inactive, and mutagenesis of Arg176 and Lys239 severely affected catalysis. The Asp154→Arg and Asp154→Glu mutant enzymes were only moderately impaired in respect of catalysis. The results strongly indicate that Arg236, which is sandwiched between Arg176 and Lys239, would initiate the reaction upon enzyme—substrate interaction, through the abstraction of the proton at C5 of the galacturonopyranose ring. The positively charged residues Arg176 and Lys239 are responsible for lowering the pKa of Arg236. Arg176 and Lys239 are maintained in a charged state by interacting with Asp154 or bulk solvent respectively. The deprotonation of the Asp186—Asp221 pair was proposed to be responsible for a pH-driven conformational change of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677—689]. Substitution of Asp186 and Asp221 by Asn186 and Asn221 was expected to stabilize the enzyme. However, the Asp186→Asn/Asp221→Asn enzyme appeared less stable than the wild-type enzyme, even at pH6.0, as evidenced by fluorescence studies. This demonstrates that the pH-dependent conformational change is not driven by deprotonation of the Asp186—Asp221 pair.

Block of Human Heart hH1 Sodium Channels by the Enantiomers of Bupivacaine

2000 ◽  
Vol 93 (4) ◽  
pp. 1022-1033 ◽  
Author(s):  
Carla Nau ◽  
Sho-Ya Wang ◽  
Gary R. Strichartz ◽  
Ging Kuo Wang
Keyword(s):  
Human Heart ◽  
Point Mutations ◽  
Cell Voltage ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Amino Acid Residues ◽  
Na Channels ◽  
State Dependent ◽  
Positively Charged

Background S(-)-bupivacaine reportedly exhibits lower cardiotoxicity but similar local anesthetic potency compared with R(+)-bupivacaine. The bupivacaine binding site in human heart (hH1) Na+ channels has not been studied to date. The authors investigated the interaction of bupivacaine enantiomers with hH1 Na+ channels, assessed the contribution of putatively relevant residues to binding, and compared the intrinsic affinities to another isoform, the rat skeletal muscle (mu1) Na+ channel. Methods Human heart and mu1 Na+ channel alpha subunits were transiently expressed in HEK293t cells and investigated during whole cell voltage-clamp conditions. Using site-directed mutagenesis, the authors created point mutations at positions hH1-F1760, hH1-N1765, hH1-Y1767, and hH1-N406 by introducing the positively charged lysine (K) or the negatively charged aspartic acid (D) and studied their influence on state-dependent block by bupivacaine enantiomers. Results Inactivated hH1 Na+ channels displayed a weak stereoselectivity with a stereopotency ratio (+/-) of 1.5. In mutations hH1-F1760K and hH1-N1765K, bupivacaine affinity of inactivated channels was reduced by approximately 20- to 40-fold, in mutation hH1-N406K by approximately sevenfold, and in mutations hH1-Y1767K and hH1-Y1767D by approximately twofold to threefold. Changes in recovery of inactivated mutant channels from block paralleled those of inactivated channel affinity. Inactivated hH1 Na+ channels exhibited a slightly higher intrinsic affinity than mu1 Na+ channels. Conclusions Differences in bupivacaine stereoselectivity and intrinsic affinity between hH1 and mu1 Na+ channels are small and most likely of minor clinical relevance. Amino acid residues in positions hH1-F1760, hH1-N1765, and hH1-N406 may contribute to binding of bupivacaine enantiomers in hH1 Na+ channels, whereas the role of hH1-Y1767 remains unclear.

scholarly journals Sequence and Structure of Human Rhinoviruses Reveal the Basis of Receptor Discrimination

2003 ◽  
Vol 77 (12) ◽  
pp. 6923-6930 ◽  
Author(s):  
Marketa Vlasak ◽  
Soile Blomqvist ◽  
Tapani Hovi ◽  
Elizabeth Hewat ◽  
Dieter Blaas
Keyword(s):  
Low Density Lipoprotein ◽  
Three Dimensional ◽  
Density Lipoprotein ◽  
Major Group ◽  
Amino Acid Residues ◽  
X Ray ◽  
Vldl Receptor ◽  
Capsid Protein Vp1 ◽  
Three Dimensional Models ◽  
Positively Charged

ABSTRACT The sequences of the capsid protein VP1 of all minor receptor group human rhinoviruses were determined. A phylogenetic analysis revealed that minor group HRVs were not more related to each other than to the nine major group HRVs whose sequences are known. Examination of the surface exposed amino acid residues of HRV1A and HRV2, whose X-ray structures are available, and that of three-dimensional models computed for the remaining eight minor group HRVs indicated a pattern of positively charged residues within the region, which, in HRV2, was shown to be the binding site of the very-low-density lipoprotein (VLDL) receptor. A lysine in the HI loop of VP1 (K224 in HRV2) is strictly conserved within the minor group. It lies in the middle of the footprint of a single repeat of the VLDL receptor on HRV2. Major group virus serotypes exhibit mostly negative charges at the corresponding positions and do not bind the negatively charged VLDL receptor, presumably because of charge repulsion.

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 Peptide recognition and dephosphorylation by the vaccinia VH1 phosphatase

2020 ◽  
Author(s):  
Bryan M. Zhao ◽  
Megan Hogan ◽  
Michael S Lee ◽  
Beverly K. Dyas ◽  
Robert G. Ulrich
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Catalytic Site ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Host Cell Proteins ◽  
Enzyme Substrate ◽  
Dual Specificity ◽  
Contact Residues

ABSTRACTThe VH1 protein encoded by the highly conserved H1 locus of orthopoxviruses is a dual-specificity phosphatase (DUSPs) that hydrolyzes phosphate groups from phosphorylated tyrosine, serine, and threonine residues of viral and host cell proteins. Because the DUSP activities are required for virus replication, VH1 is a prime target for the development of therapeutic inhibitors. However, the presentation of a shallow catalytic site has thwarted all drug development efforts. As an alternative to direct targeting of catalytic pockets, we describe surface contacts between VH1 and substrates that are essential for full activity and provide a new pathway for developing inhibitors of protein-protein interactions. Critical amino acid residues were manipulated by site-directed mutagenesis of VH1, and perturbation of peptide substrate interactions based on these mutations were assessed by high-throughput assays that employed surface plasmon resonance and phosphatase activities. Two positively-charged residues (Lys-20 and Lys-22) and the hydrophobic side chain of Met-60 appear to orient the polarity of the pTyr peptide on the VH1 surface, while additional amino acid residues that flank the catalytic site contribute to substrate recognition and productive dephosphorylation. We propose that the enzyme-substrate contact residues described here may serve as molecular targets for the development of inhibitors that specifically block VH1 catalytic activity and thus poxvirus replication.

scholarly journals Site-directed mutagenesis of β-lactamase I. Single and double mutants of Glu-166 and Lys-73

1990 ◽  
Vol 272 (3) ◽  
pp. 613-619 ◽  
Author(s):  
R M Gibson ◽  
H Christensen ◽  
S G Waley
Keyword(s):  
Rate Constants ◽  
Double Mutant ◽  
Enzyme Mechanism ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Burst Kinetics ◽  
Hydrolysis Of

Two single mutants and the corresponding double mutant of beta-lactamase I from Bacillus cereus 569/H were constructed and their kinetics investigated. The mutants have Lys-73 replaced by arginine (K73R), or Glu-166 replaced by aspartic acid (E166D), or both (K73R + E166D). All four rate constants in the acyl-enzyme mechanism were determined for the E166D mutant by the methods described by Christensen, Martin & Waley [(1990) Biochem. J. 266, 853-861]. Both the rate constants for acylation and deacylation for the hydrolysis of benzylpenicillin were decreased about 2000-fold in this mutant. In the K73R mutant, and in the double mutant, the rate constants for acylation were decreased about 100-fold and 10,000-fold respectively. All three mutants also had lowered values for the rate constants for the formation and dissociation of the non-covalent enzyme-substrate complex. The specificities of the mutants did not differ greatly from those of wild-type beta-lactamase, but the hydrolysis of cephalosporin C by the K73R mutant gave ‘burst’ kinetics.

Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase

2006 ◽  
Vol 387 (5) ◽  
pp. 515-523 ◽  
Author(s):  
Shivakumara Bheemanaik ◽  
Janusz M. Bujnicki ◽  
Valakunja Nagaraja ◽  
Desirazu N. Rao
Keyword(s):  
Dna Methyltransferase ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Three Dimensional Model ◽  
Cofactor Binding ◽  
Protein Fold Recognition ◽  
Fluorescence Measurements ◽  
Complete Disruption

AbstractKpnI DNA-(N6-adenine) methyltransferase (M.KpnI) recognises the sequence 5′-GGTACC-3′ and transfers the methyl group fromS-adenosyl-L-methionine (AdoMet) to the N6 position of the adenine residue in each strand. Earlier studies have shown that M.KpnI exists as a dimer in solution, unlike most other MTases. To address the importance of dimerisation for enzyme function, a three-dimensional model of M.KpnI was obtained based on protein fold-recognition analysis, using the crystal structures of M.RsrI and M.MboIIA as templates. Residues I146, I161 and Y167, the side chains of which are present in the putative dimerisation interface in the model, were targeted for site-directed mutagenesis. Methylation andin vitrorestriction assays showed that the mutant MTases are catalytically inactive. Mutation at the I146 position resulted in complete disruption of the dimer. The replacement of I146 led to drastically reduced DNA and cofactor binding. Substitution of I161 resulted in weakening of the interaction between monomers, leading to both monomeric and dimeric species. Steady-state fluorescence measurements showed that the wild-type KpnI MTase induces structural distortion in bound DNA, while the mutant MTases do not. The results establish that monomeric MTase is catalytically inactive and that dimerisation is an essential event for M.KpnI to catalyse the methyl transfer reaction.

Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots

2011 ◽  
Vol 437 (2) ◽  
pp. 223-230 ◽  
Author(s):  
Han-Woo Kim ◽  
Kazuhiko Ishikawa
Keyword(s):  
Enzyme Reaction ◽  
Intermediate Structure ◽  
Amino Acid Residues ◽  
Pyrococcus Horikoshii ◽  
Substrate Complex ◽  
Substrate Structure ◽  
Enzyme Substrate ◽  
Cellulose Substrates ◽  
Boat Form ◽  
Low Activity

A hyperthermophilic membrane-related β-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65–2.01 Å (1 Å=0.1 nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite −1, and the β-1,4-glucoside bond between glucose moieties is twisted between subsites −1 and +1. Subsite −2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite −1. A rare enzyme–substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme–substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.

scholarly journals Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity

2002 ◽  
Vol 365 (3) ◽  
pp. 731-738 ◽  
Author(s):  
Carlos NOVO ◽  
Sebastien FARNAUD ◽  
Renée TATA ◽  
Alda CLEMENTE ◽  
Paul R. BROWN
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Substrate Specificity ◽  
Catalytic Mechanism ◽  
Three Dimensional ◽  
Data Bank ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Three Dimensional Models

The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys166 is the nucleophile of the catalytic mechanism. A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase—fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template. The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit. Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads. Support for the structure for the P. aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated.

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