Thank God for noncompliance--this time, at least.
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Aged↗
Biomedical subjects
K M Peterson
Publications and source records attributed to K M Peterson.
Influence of Glu-376 --> Gln mutation on enthalpy and heat capacity changes for the binding of slightly altered ligands to medium chain acyl-CoA dehydrogenase.
We showed that the alpha-CH(2) --> NH substitution in octanoyl-CoA alters the ground and transition state energies for the binding of the CoA ligands to medium-chain acyl-CoA dehydrogenase (MCAD), and such an effect is caused by a small electrostatic difference between the ligands. To ascertain the extent that the electrostatic contribution of the ligand structure and/or the enzyme site environment modulates the thermodynamics of the enzyme-ligand interaction, we undertook comparative microcalorimetric studies for the binding of 2-azaoctanoyl-CoA (alpha-CH(2) --> NH substituted octanoyl-CoA) and octenoyl-CoA to the wild-type and Glu-376 --> Gln mutant enzymes. The experimental data revealed that both enthalpy (DeltaH degrees ) and heat capacity changes (DeltaC(p) degrees ) for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -21.7 +/- 0.8 kcal/mole, DeltaC(p) degrees = -0.627 +/- 0.04 kcal/mole/K) to the wild-type MCAD were more negative than those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -17.2 +/- 1.6 kcal/mole, DeltaC(p) degrees = -0.526 +/- 0.03 kcal/mole/K). Of these, the decrease in the magnitude of DeltaC(p) degrees for the binding of 2-azaoctanoyl-CoA (vis-à-vis octenoyl-CoA) to the enzyme was unexpected, because the former ligand could be envisaged to be more polar than the latter. To our further surprise, the ligand-dependent discrimination in the above parameters was completely abolished on Glu-376 --> Gln mutation of the enzyme. Both DeltaH degrees and DeltaC(p) degrees values for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -13.3 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.511 +/- 0.03 kcal/mole/K) to the E376Q mutant enzyme were found to be correspondingly identical to those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -13.2 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.520 +/- 0.02 kcal/mole/K). However, in neither case could the experimentally determined DeltaC(p) degrees values be predicted on the basis of the changes in the water accessible surface areas of the enzyme and ligand species. Arguments are presented that the origin of the above thermodynamic differences lies in solvent reorganization and water-mediated electrostatic interaction between ligands and enzyme site groups, and such interactions are intrinsic to the molecular basis of the enzyme-ligand complementarity.
Acyl Coenzyme A↗
Influence of alpha-CH-->NH substitution in C8-CoA on the kinetics of association and dissociation of ligands with medium chain acyl-CoA dehydrogenase.
We previously reported that the kinetic profiles for the association and dissociation of functionally diverse C(8)-CoA-ligands, viz., octanoyl-CoA (substrate), octenoyl-CoA (product), and octynoyl-CoA (inactivator) with medium chain acyl-CoA dehydrogenase (MCAD), were essentially identical, suggesting that the protein conformational changes played an essential role during ligand binding and/or catalysis [Peterson, K. L., Sergienko, E. E., Wu, Y., Kumar, N. R., Strauss, A. W., Oleson, A. E., Muhonen, W. W., Shabb, J. B., and Srivastava, D. K. (1995) Biochemisry 34, 14942-14953]. To ascertain the structural basis of the above similarity, we investigated the kinetics of association and dissociation of alpha-CH-->NH-substituted C(8)-CoA, namely, 2-azaoctanoyl-CoA, with the recombinant form of human liver MCAD. The rapid-scanning and single wavelength stopped-flow data for the binding of 2-azaoctanoyl-CoA to MCAD revealed that the overall interaction proceeds via two steps. The first (fast) step involves the formation of an enzyme-ligand collision complex (with a dissociation constant of K(c)), followed by a slow isomerization step (with forward and reverse rate constants of k(f) and k(r), respectively) with concomitant changes in the electronic structure of the enzyme-bound FAD. Since the latter step involves a concurrent change in the enzyme's tryptophan fluorescence, it is suggested that the isomerization step is coupled to the changes in the protein conformation. Although the overall binding affinity (K(d)) of the enzyme-2-azaoctanoyl-CoA complex is similar to that of the enzyme-octenoyl-CoA complex, their microscopic equilibria within the collision and isomerized complexes show an opposite relationship. These results coupled with the isothermal titration microcalorimetric studies lead to the suggestion that the electrostatic interaction within the enzyme site phase modulates the microscopic steps, as well as their corresponding ground and transition states, during the course of the enzyme-ligand interaction.
Acyl Coenzyme A↗
Energetic consequences of accommodating a bulkier ligand at the active site of medium chain acyl-CoA dehydrogenase by creating a complementary enzyme site cavity.
The substitution of the C=O by the C=S group in 2-azaoctanoyl-CoA increases the volume of the ligand by 11 A(3), and the excision of a methylene group from Glu-376, via Glu-376 --> Asp (E376D) mutation in medium chain acyl-CoA dehydrogenase (MCAD), creates a complementary cavity of 18 A(3) dimension, just opposite to the ligand's carbonyl group. We investigated whether the newly created cavity would facilitate accommodation of the bulkier (C=O --> C=S substituted) ligand within the active site of the enzyme. To ascertain this, we determined the binding affinity and kinetics of association and dissociation of 2-azaoctanoyl-CoA and the C=O --> C=S substituted ligand, 2-azadithiooctanoyl-CoA, involving the wild-type and Glu-376 --> Asp mutant enzymes. The experimental data revealed that the binding of 2-azadithiooctanoyl-CoA to the wild-type enzyme was energetically unfavorable as compared to 2-azaoctanoyl-CoA. However, such an energetic constraint was alleviated for the binding of the former ligand to the E376D mutant enzyme site. A detailed account of the free energy and enthalpic profiles for the binding of 2-azaoctanoyl-CoA and 2-azadithiooctanoyl-CoA to the wild-type and Glu-376 --> Asp mutant enzymes throws light on the flexibility of the enzyme site cavity in stabilizing the ground and transition states of the enzyme-ligand complexes.
Acyl Coenzyme A↗
Human ATP-binding cassette transporter 1 (ABC1): genomic organization and identification of the genetic defect in the original Tangier disease kindred.
Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.
ATP Binding Cassette Transporter 1↗
Thermodynamics of ligand binding and catalysis in human liver medium-chain acyl-CoA dehydrogenase: comparative studies involving normal and 3'-dephosphorylated C8-CoAs and wild-type and Asn191 --> Ala (N191A) mutant enzymes.
Following our demonstration that the terminal 3'-phosphate group of acyl-CoA substrates (which is confined to the exterior of the protein structure, and is fully exposed to the outside solvent environment) exhibits a functional role in the recombinant human liver medium-chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction [Peterson, K. L., and Srivastava, D. K. (1997) Biochem. J. 325, 751-760], we became interested in delineating its thermodynamic contribution in stabilizing the "ground" and "transition" state structures during enzyme catalysis. Since the 3'-phosphate group of the coenzyme A thiolester has the potential to form a hydrogen bond with the side chain group of Asn-191, these studies were performed utilizing both normal and 3'-dephosphorylated forms of octanoyl-CoA and octenoyl-CoA (cumulatively referred to as C8-CoA) as the physiological substrate and product of the enzyme, respectively, as well as utilizing wild-type and Asn191 --> Ala (N191A) site-specific mutant enzymes. The experimental data revealed that the enthalpic contribution of the 3'-phosphate group was similar in both ground and transition states, and was primarily derived from the London-van der Waals interactions (between the 3'-phosphate group of C8-CoA and the surrounding protein moiety), rather than from the potential hydrogen bonding. The temperature dependence of DeltaH degrees for the binding of octenoyl-CoA and 3'-dephosphooctenoyl-CoA revealed that the deletion of the 3'-phosphate group from octenoyl-CoA increased the magnitude of the heat capacity changes (DeltaCp degrees) from -0.53 to -0.59 kcal mol-1 K-1. Although the latter effect could be attributed to an increase in the relative hydrophobicity of the ligand, the experimentally observed DeltaCp degrees's (for either of the ligands) could not be predicted on the basis of the changes in the solvent-accessible surface areas of the enzyme and ligand species. These coupled with the fact that the DeltaCp degrees for the binding of octenoyl-CoA to pig kidney MCAD (which is believed to be structurally identical to human liver MCAD) is only -0.37 kcal mol-1 K-1 [Srivastava, D. K., Wang, S., and Peterson, K. L. (1997) Biochemistry 36, 6359-6366] prompt us to question the reliability of predicting the DeltaCp degrees values of the enzyme-ligand complexes from their X-ray crystallographic data. Arguments are presented that certain intrinisic limitations of the crystallographic data preclude kinetic and thermodynamic predictions about the enzyme-ligand complexes and enzyme catalysis.
Acyl Coenzyme A↗
Simultaneous detection of multiple analytes using Copalis technology: a reduction to practice.
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Chemistry, Clinical↗
Cloning and nucleotide sequence analysis of a Brucella abortus gene encoding an 18 kDa immunoreactive protein.
A DNA fragment encoding an approximately 18 kDa protein from Brucella abortus strain 2308 was cloned and expressed in Escherichia coli. This recombinant protein, designated BA18K, reacted in Western blot analysis with sera obtained from experimentally and naturally infected animals including mice, goats, dogs and humans. Restriction enzyme analysis of the plasmid (pBA28) encoding BA18K revealed the presence of an approximately 8.7 kbp Sau3A genomic DNA fragment within the vector and subsequent subcloning and Western blot analysis limited the region encoding BA18K to an approximately 3.0 kbp Pst 1 DNA fragment. DNA sequence analysis of this region identified an open reading frame capable of encoding a protein of 177 amino acids with a predicted relative molecular mass of 17529. Comparison of the deduced amino acid sequence of BA18K with those in the protein sequence databases yielded no homology with previously described proteins from other bacterial genera. These searches did, however, indicate that BA18K is identical to the previously described outer membrane protein (OMP) from B. abortus strain 544 designated Omp 19.
Amino Acid Sequence↗
A putative integrase gene defines the distal end of a large cluster of ToxR-regulated colonization genes in Vibrio cholerae.
A large cluster of virulence genes encoding proteins involved in Vibrio cholerae accessory colonization factor (ACF) expression and toxin-coregulated pilus (TCP) biogenesis is flanked by sequences that resemble bacteriophage attachment (att) half-sites. Adjacent to the attL-like site is a gene (int) that encodes a protein related to the integrase family of site-specific recombinases. The putative vibrio integrase appears to be most closely related to the Escherichia coli cryptic prophage (CP4-57) integrase protein (52% identity, 73% similarity). Genomic analysis of numerous V. cholerae strains (O1, non-O1 and O139) revealed that only vibrios capable of causing epidemic Asiatic cholera possess the TCP-ACF colonization gene cluster in association with the integrase. The fact that the integrase gene is absent in avirulent strains suggests that epidemic strains of V. cholerae obtained the TCP-ACF colonization gene cluster via horizontal transfer.
Amino Acid Sequence↗
Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.
Four new antibiotic-resistant derivatives of the broad-host-range (bhr) cloning vector pBBR1MCS have been constructed. These new plasmids have several advantages over many of the currently available bhr vectors in that: (i) they are relatively small (< 5.3 kb), (ii) they possess an extended multiple cloning site (MCS), (iii) they allow direct selection of recombinant plasmid molecules in Escherichia coli via disruption of the LacZ alpha peptide, (iv) they are mobilizable when the RK2 transfer functions are provided in trans and (v) they are compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons.
Bacteria↗
Isolation and characterization of the Vibrio cholerae acfA gene, required for efficient intestinal colonization.
The nucleotide sequence of the Vibrio cholerae acfA gene (encoding an accessory colonization factor) has been determined. Sequence analysis revealed the presence of an open reading frame of 215 amino acids with a characteristic signal peptidase I (SPI) cleavage site at the N terminus. Electrophoretic analysis of proteins synthesized by Escherichia coli cells, following T7 promoter/RNA polymerase-directed expression of acfA, revealed a 23-kDa protein corresponding to the mature form of AcfA. The T7 expression system also showed that, in the presence of known SPI inhibitors, a 25-kDa unprocessed form of AcfA is produced.
Amino Acid Sequence↗
Isolation and characterization of a Vibrio cholerae gene (tagA) that encodes a ToxR-regulated lipoprotein.
The Vibrio cholerae (Vc) gene (tagA) coding for the TagA lipoprotein has been isolated. Sequencing of tagA revealed the presence of an open reading frame (ORF) of 568 amino acids with a characteristic signal peptidase II cleavage site at the N terminus. Electrophoretic analysis of proteins synthesized by Escherichia coli (Ec) cells following T7 promoter/RNA polymerase-directed expression of tagA, revealed a closely migrating doublet of proteins corresponding to two species of TagA. Computer-generated alignment algorithms predict that a homology exists between Vc TagA and Ec K99 fimbriae biogenesis determinant FanD.
Algorithms↗
In vivo and in vitro stability of the broad-host-range cloning vector pBBR1MCS in six Brucella species.
Previous studies have shown that the broad-host-range plasmid pBBR1MCS can be used for genetic complementation in Brucella abortus. To extend these observations, the in vivo and in vitro stability of pBBR1MCS was evaluated in the six currently recognized species of the genus Brucella. pBBR1MCS was readily introduced into all of the strains tested by electroporation and was stably maintained in broth cultures without antibiotic selection during five serial passages over a 10-day period. Furthermore, isolates of all six Brucella strains containing pBBR1MCS obtained from the spleens of BALB/c mice 1 week postinfection maintained the plasmid. Although pBBR1MCS maintains the mobilization locus present in the parental plasmid pBBR1CM, attempts to detect transfer of pBBR1MCS between Brucella strains by conjugation were unsuccessful. These results demonstrate the in vitro and in vivo stability of pBBR1MCS in Brucella spp. and reinforce the usefulness of this cloning vector for the genetic analysis of these organisms.
Animals↗
The Vibrio cholerae hlyC gene encodes a protein that is related to lipases of pseudomonas species.
The nucleotide sequence of the Vibrio cholerae N16961 hlyC gene was determined. The hlyC gene encompasses 513 nucleotides that are predicted to encode a 171-amino acid protein with a calculated molecular weight of 18.2 kDa. The predicted HlyC protein contains a region that is 93.5% similar to the substrate-binding/catalytic domain of the Pseudomonas species triacylglycerol acylhydrolase (lipase). The proposed catalytic serine residue is also conserved in the HlyC protein. The contribution of the putative HlyC lipase to the physiology of V. cholerae is currently under investigation.
Amino Acid Sequence↗
Identification of a ToxR-activated gene, tagE, that lies within the accessory colonization factor gene cluster of Vibrio cholerae O395.
The nucleotide (nt) sequence has been determined for a Vibrio cholerae ToxR-activated gene designated tagE that is located within a cluster of genes required for efficient intestinal colonization. The tagE gene encompasses 909 nt and is predicted to encode a 303-amino-acid (aa) protein with an estimated molecular mass of 34,468 Da. Computer-assisted similarity searches revealed that TagE possesses aa sequence similarity with Escherichia coli OrfU and Staphylococcus simulans lysostaphin, two proteins that are involved in cell-wall biosynthesis and peptidoglycan degradation, respectively. The role, if any, that TagE plays in the accessory colonization factor phenotype is currently under investigation.
Amino Acid Sequence↗
Identification of a Vibrio cholerae ToxR-activated gene (tagD) that is physically linked to the toxin-coregulated pilus (tcp) gene cluster.
The toxin-coregulated pilus (TCP)-encoding gene cluster (tcp) specifies a type-IV pilus that is a major colonization determinant of Vibrio cholerae. We have identified a gene 200 bp upstream from the tcp cluster that requires ToxR for expression. We have designated this gene tagD (ToxR-activated gene) and have shown that tagD is encoded on a 600-nt transcript. The deduced tagD product is a 164-amino-acid polypeptide (20 kDa). Interestingly, TagD shares a high degree of similarity to a protein of Streptococcus sanguis 12 that is thought to play a role in fimbriae synthesis or assembly. The high degree of similarity between tagD and the Ss 12 protein provides preliminary evidence that tagD represents an additional member of the tcp cluster.
Amino Acid Sequence↗
Sequence analysis of the Vibrio cholerae acfD gene reveals the presence of an overlapping reading frame, orfZ, which encodes a protein that shares sequence similarity to the FliA and FliC products of Salmonella.
The nucleotide (nt) sequence of the Vibrio cholerae acfD gene (encoding an accessory colonization factor) has been determined. The acfD gene encompasses 254 nt that are predicted to encode an 88-amino-acid (aa) protein. Additionally, an open reading frame of 184 aa, designated orfZ, was detected that overlaps the 3' end of acfD by 45 nt. Computer-assisted homology searches revealed that OrfZ possesses aa sequence similarity to the C terminus of the fliC product of Salmonella muenchen and S. rubislaw. Additionally, OrfZ shares limited aa sequence similarity to a region of the S. typhimurium fliA product. Interestingly, a V. cholerae acfD::TnphoA mutant demonstrates reduced motility and an altered swarming phenotype in semi-solid media when compared to wild-type V. cholerae. The possibility that the reduced motility and altered morphology result from polar effects on orfZ expression is currently being investigated.
Amino Acid Sequence↗
The determined state of white expression in the Drosophila eye is modified by zeste1 in the wzm family of mutants.
Analysis of the whitezeste mottled (wzm) mutant family suggests that the zeste gene product functions in establishing and stabilizing a transcriptionally active chromatin domain for white locus expression. The z1 mutation reduces expression of paired or proximate copies of white, while single or unpaired copies maintain wild-type levels of expression. The wzm mutation, caused by the insertion of the retrotransposon BEL into the 5' intron of white, alters the zeste-white interaction to produce a mottled eye phenotype in hemizygous z1 wzm males. We have determined the molecular structure of four wzm derivatives. wzl results from the insertion of an additional transposable element into the 5' regulatory region of white. wzvl is a deletion of sequences upstream of the white locus. Two others, whalo and wcres, result from the transposition of wzm plus the entire verticals-roughest region into heterochromatin near the tip of chromosome 3L. They variegate for roughest but not for white; rather, the z1 effect on wzm now causes white expression to become non-autonomous and non-clonal. The analysis of these five mutations shows that the neomorphic zeste1 product, in combination with structural changes imposed by transposons and intercalary heterochromatin, modifies the determination and stability of white expression. We propose that the normal zeste product functions as part of a complex that stimulates transcription by changing chromatin conformation to establish and maintain transcriptionally active domains. The unpairing of homologs is proposed to be one of the initial results of conformational change, providing an explanation for the role of zeste in transvection.
Alleles↗