Crystal engineering: a case study using the 24 kDa fragment of the DNA gyrase B subunit from Escherichia coli

1999 ◽  
Vol 55 (9) ◽  
pp. 1623-1625 ◽  
Author(s):  
Allan D'Arcy ◽  
Martine Stihle ◽  
Dirk Kostrewa ◽  
Glenn Dale
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Crystal Engineering ◽  
Dna Gyrase ◽  
Site Directed Mutagenesis ◽  
Crystal Quality ◽  
Single Amino Acid ◽  
Wild Type ◽  
B Subunit

Site-directed mutagenesis was used to determine the efficacy of changing surface residues to improve crystal quality. Nine mutants of the 24 kDa fragment of the Escherichia coli DNA gyrase B subunit were produced, changing residues on the protein's surface. The mutations changed either the charge or the polarity of the wild-type amino acid. It was found that single amino-acid changes on the surface could have a dramatic effect on the crystallization properties of the protein and generally resulted in an improvement in the number of crystal-screen hits as well as an improvement in crystal quality. It is concluded that crystal engineering is a valuable tool for protein crystallography.

Crystal engineering: deletion mutagenesis of the 24 kDa fragment of the DNA gyrase B subunit from Staphylococcus aureus

1999 ◽  
Vol 55 (9) ◽  
pp. 1626-1629 ◽  
Author(s):  
Glenn E. Dale ◽  
Dirk Kostrewa ◽  
Bernard Gsell ◽  
Martin Stieger ◽  
Allan D'Arcy
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Crystal Engineering ◽  
Dna Gyrase ◽  
Wild Type ◽  
Scanning Calorimetry ◽  
Mutant Proteins ◽  
B Subunit ◽  
Deletion Mutagenesis ◽  
Single Well

The 24 kDa fragment of DNA gyrase B from Staphylococcus aureus was expressed in Escherichia coli and purified for crystallization. Crystals of the wild-type protein grew in the presence of cyclothialidine but proved difficult to reproduce. In order to improve the crystallization, the flexible regions of the protein were deleted by mutagenesis. The mutant proteins were analyzed by differential scanning calorimetry and the most stable mutants produced crystals. It was possible to reproducibly grow in the microbatch system single well defined crystals which belonged to the space group C2 and diffracted isotropically to approximately 2 Å resolution.

scholarly journals Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity

1989 ◽  
Vol 86 (17) ◽  
pp. 6577-6581 ◽  
Author(s):  
T W Seeley ◽  
L Grossman
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Excision Repair ◽  
Site Directed Mutagenesis ◽  
Sequence Motif ◽  
Amino Acid Side Chain ◽  
Uv Resistance ◽  
Wild Type ◽  
General Applicability ◽  
Repair Capacity

The Escherichia coli UvrB protein possesses an amino acid sequence motif common to many ATPases. The role of this motif in UvrB has been investigated by site-directed mutagenesis. Three UvrB mutants, with amino acid replacements at lysine-45, failed to confer UV resistance when tested in the UV-sensitive strain N364 (delta uvrB), while five other mutants constructed near this region of UvrB confer wild-type levels of UV resistance. Because even the conservative substitution of arginine for lysine-45 in UvrB results in failure to confer UV resistance, we believe we have identified an amino acid side chain in UvrB essential to nucleotide excision repair in E. coli. The properties of two purified mutant UvrB proteins, lysine-45 to alanine (K45A) and asparagine-51 to alanine (N51A), were analyzed in vitro. While the K45A mutant is fully defective in incision of UV-irradiated DNA, K45A is capable of interaction with UvrA in forming an ATP-dependent nucleoprotein complex. The K45A mutant, however, fails to activate the characteristic increase in ATPase activity observed with the wild-type UvrB in the presence of UvrA and DNA. From these results we conclude that there is a second nucleotide-dependent step in incision following initial complex formation, which is defective in the K45A mutant. This experimental approach may prove of general applicability in the study of function and mechanism of other ATPase motif proteins.

scholarly journals A Single Amino Acid Substitution in a Mannosyltransferase, WbdA, Converts the Escherichia coli O9 Polysaccharide into O9a: Generation of a New O-Serotype Group

2000 ◽  
Vol 182 (9) ◽  
pp. 2567-2573 ◽  
Author(s):  
Nobuo Kido ◽  
Hidemitsu Kobayashi
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Chimeric Genes ◽  
Functional Studies

ABSTRACT wbdA is a mannosyltransferase gene that is involved in synthesis of the Escherichia coli O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1. The equivalent structural O polysaccharide in the E. coli O9 andKlebsiella O3 strains is 2-αMan-1,2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1, with an excess of one mannose in the 1,2 linkage. We have cloned wbdAgenes from these O9 and O3 strains and shown by genetic and functional studies that wbdA is the only gene determining the O-polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we showed that a single amino acid substitution, C55R, in WbdA of E. coli O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to be conserved in other E. coli O9a strains. The reverse substitution, R55C, in WbdA of E. coli O9a resulted in lipopolysaccharide synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in WbdA is required for conversion from O9a to O9.

scholarly journals Structural characterization of human aryl sulphotransferases

1999 ◽  
Vol 337 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Lulu A. BRIX ◽  
Ronald G. DUGGLEBY ◽  
Andrea GAEDIGK ◽  
Michael E. McMANUS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Change ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Wild Type ◽  
Substrate Specificities ◽  
Loop Region ◽  
Single Amino Acid Change

Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greater than 90% sequence identity, but vary markedly in their ability to catalyse the sulphonation of dopamine and p-nitrophenol. In order to investigate the amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes two regions of high sequence divergence, termed Regions A (amino acids 44–107) and B (amino acids 132–164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to change 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specificity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single amino acid change. However, compared with wild-type HAST1, a number of the mutations resulted in interference with substrate binding, as shown by elevated Ki values towards the co-substrate 3´-phosphoadenosine 5´-phosphosulphate, and in some cases loss of activity towards dopamine. These findings suggest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopamine, whereas a single amino acid at position 146 determines p-nitrophenol affinity. A HAST1 mutant was constructed to express a protein with four amino acids deleted (P87–P90). These amino acids were hypothesized to correspond to a loop region in close proximity to the substrate-binding pocket. Interestingly, the protein showed substrate specificities more similar to wild-type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.

scholarly journals Molecular and Functional Analyses of Kunjin Virus Infectious cDNA Clones Demonstrate the Essential Roles for NS2A in Virus Assembly and for a Nonconservative Residue in NS3 in RNA Replication

2003 ◽  
Vol 77 (14) ◽  
pp. 7804-7813 ◽  
Author(s):  
Wen Jun Liu ◽  
Hua Bo Chen ◽  
Alexander A. Khromykh
Keyword(s):  
Amino Acid ◽  
Virus Assembly ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Common Amino Acid

ABSTRACT A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by ∼100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an ∼5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type Ile residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

scholarly journals Overproduction of l-Cysteine andl-Cystine by Escherichia coli Strains with a Genetically Altered Serine Acetyltransferase

1998 ◽  
Vol 64 (5) ◽  
pp. 1607-1611 ◽  
Author(s):  
Shigeru Nakamori ◽  
Shin-ichiro Kobayashi ◽  
Chitose Kobayashi ◽  
Hiroshi Takagi
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Feedback Inhibition ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Serine Acetyltransferase ◽  
Methionine Residue ◽  
Cysteine Desulfhydrase ◽  
K 12

ABSTRACT Organisms that overproduced l-cysteine andl-cystine from glucose were constructed by usingEscherichia coli K-12 strains. cysE genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by l-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered cysE genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by l-cysteine. At the same time, these transformants produced approximately 200 mg ofl-cysteine plus l-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetyltransferase gene. However, the production ofl-cysteine and l-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production ofl-cysteine plus l-cystine was markedly increased compared to production in JM39.

Effects of amino acid substitutions at the active site in Escherichia coli beta-galactosidase.

Genetics ◽  
1988 ◽  
Vol 120 (3) ◽  
pp. 637-644
Author(s):  
C G Cupples ◽  
J H Miller
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Lacz Gene ◽  
Wild Type ◽  
Mutant Proteins ◽  
Beta Galactosidase

Abstract Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme beta-galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac-. Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1% wild-type activity. His-464 and Met-3 were replaced with 11 and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac+. The results support previous evidence that Glu-461 and Tyr-503 are essential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an f1 bacteriophage containing the complete lacZ gene on a single EcoRI fragment.

scholarly journals The molecular mechanism for the spectral shifts between vertebrate ultraviolet- and violet-sensitive cone visual pigments

2002 ◽  
Vol 367 (1) ◽  
pp. 129-135 ◽  
Author(s):  
Jill A. COWING ◽  
Subathra POOPALASUNDARAM ◽  
Susan E. WILKIE ◽  
Phyllis R. ROBINSON ◽  
James K. BOWMAKER ◽  
...  
Keyword(s):  
Amino Acid ◽  
Short Wave ◽  
Visual Pigments ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Wild Type ◽  
Sequence Comparisons ◽  
Molecular Identity ◽  
Spectral Shifts ◽  
Mammalian Lineage

The short-wave-sensitive (SWS) visual pigments of vertebrate cone photoreceptors are divided into two classes on the basis of molecular identity, SWS1 and SWS2. Only the SWS1 class are present in mammals. The SWS1 pigments can be further subdivided into violet-sensitive (VS), with λmax (the peak of maximal absorbance) values generally between 400 and 430nm, and ultraviolet-sensitive (UVS), with a λmax<380nm. Phylogenetic evidence indicates that the ancestral pigment was UVS and that VS pigments have evolved separately from UVS pigments in the different vertebrate lineages. In this study, we have examined the mechanism of evolution of VS pigments in the mammalian lineage leading to present day ungulates (cow and pig). Amino acid sequence comparisons of the UVS pigments of teleost fish, amphibia, reptiles and rodents show that site 86 is invariably occupied by Phe but is replaced in bovine and porcine VS pigments by Tyr. Using site-directed mutagenesis of goldfish UVS opsin, we have shown that a Phe-86→Tyr substitution is sufficient by itself to shift the λmax of the goldfish pigment from a wild-type value of 360nm to around 420nm, and the reverse substitution of Tyr-86—Phe into bovine VS opsin produces a similar shift in the opposite direction. The substitution of this single amino acid is sufficient to account therefore for the evolution of bovine and porcine VS pigments. The replacement of Phe with polar Tyr at site 86 is consistent with the stabilization of Schiff-base protonation in VS pigments and the absence of protonation in UVS pigments.

scholarly journals Conversion of NfsA, the Major Escherichia coliNitroreductase, to a Flavin Reductase with an Activity Similar to That of Frp, a Flavin Reductase in Vibrio harveyi, by a Single Amino Acid Substitution

1998 ◽  
Vol 180 (2) ◽  
pp. 422-425 ◽  
Author(s):  
Shuhei Zenno ◽  
Toshiro Kobori ◽  
Masaru Tanokura ◽  
Kaoru Saigo
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Hydrogen Bonds ◽  
Amino Acid Sequence ◽  
Amino Acid Substitution ◽  
Vibrio Harveyi ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Flavin Reductase ◽  
Wild Type

ABSTRACT NfsA is the major oxygen-insensitive nitroreductase ofEscherichia coli, similar in amino acid sequence to Frp, a flavin reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroreductase into a flavin reductase that is as active as the authentic Frp and a tartrazine reductase that is 30-fold more active than wild-type NfsA.

scholarly journals Two “Wild-Type” Variants of Escherichia coli σ70: Context-Dependent Effects of the Identity of Amino Acid 149

2002 ◽  
Vol 184 (4) ◽  
pp. 1192-1195 ◽  
Author(s):  
Nicole E. Baldwin ◽  
Andrea McCracken ◽  
Alicia J. Dombroski
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Amino Acid Substitution ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Wild Type ◽  
Context Dependent

ABSTRACT The identity of amino acid 149 of Escherichia coli σ70 has been reported variably as either arginine or aspartic acid. We show that the behavior of both a region 1.2 deletion and a single-amino-acid substitution at position 122 are greatly affected by the identity of amino acid 149.

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