scholarly journals Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds

1997 ◽  
Vol 327 (2) ◽  
pp. 537-544 ◽  
Author(s):  
F. Catherine OLIVER ◽  
Sandeep MODI ◽  
U. William PRIMROSE ◽  
Lu-Yun LIAN ◽  
C. K. Gordon ROBERTS
Keyword(s):  
Fatty Acids ◽  
Bacillus Megaterium ◽  
Three Dimensional ◽  
Pair Interaction ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Steady State Kinetics ◽  
In The Wild ◽  
Cytochrome P 450

Oligonucleotide-directed mutagenesis has been used to replace arginine-47 with glutamate in cytochrome P-450 BM3 from Bacillus megaterium and in its haem domain. The mutant has been characterized by sequencing, mass spectrometry, steady-state kinetics and by optical and NMR measurements of substrate binding. The mutant retains significant catalytic activity towards C12-C16 fatty acids, catalysing hydroxylation in the same (ω-1, ω-2, ω-3) positions with kcat/Km values a factor of 14-21 lower. C12-C16 alkyl trimethylammonium compounds are relatively poor substrates for the wild-type enzyme, but are efficiently hydroxylated by the arginine-47 → glutamate mutant at the ω-1, ω-2 and ω-3 positions, with kcat values of up to 19 s-1. Optical spectroscopy shows that the binding of the C14 and C16 alkyl trimethylammonium compounds to the mutant is similar to that of the corresponding fatty acids to the wild-type enzyme. Paramagnetic relaxation measurements show that laurate binds to the ferric state of the mutant in a significantly different position, 1.5 Å closer to the iron, than seen in the wild-type, although this difference is much smaller (~ 0.2 Å) in the ferrous state of the complex. The binding of a substrate having the same charge as residue 47 to the ferric state of the enzyme is roughly ten times weaker than that of a substrate having the opposite charge (and thus is able to make an ion-pair interaction with this residue). The results are discussed in the light of the three-dimensional structure of the enzyme.

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

2001 ◽  
Vol 356 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Ricardo FRANCO ◽  
Alice S. PEREIRA ◽  
Pedro TAVARES ◽  
Arianna MANGRAVITA ◽  
Michael J. BARBER ◽  
...  
Keyword(s):  
Absorption Spectra ◽  
Catalytic Mechanism ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Enzymic Activity ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

scholarly journals Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

1999 ◽  
Vol 181 (14) ◽  
pp. 4397-4403 ◽  
Author(s):  
Casper Jørgensen ◽  
Gert Dandanell
Keyword(s):  
Amino Acids ◽  
Purine Nucleoside Phosphorylase ◽  
Mutational Analysis ◽  
Regulatory Protein ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Wild Type ◽  
Isolation And Characterization ◽  
In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

Concerted action of two subunits of the functional dimer ofShewanella oneidensisMR-1 uridine phosphorylase derived from a comparison of the C212S mutant and the wild-type enzyme

2016 ◽  
Vol 72 (2) ◽  
pp. 203-210 ◽  
Author(s):  
T. N. Safonova ◽  
N. N. Mordkovich ◽  
V. P. Veiko ◽  
N. A. Okorokova ◽  
V. A. Manuvera ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Shewanella Oneidensis ◽  
Dimensional Structure ◽  
Free Form ◽  
Uridine Phosphorylase ◽  
Wild Type ◽  
Concerted Action ◽  
Wild Type Enzyme ◽  
Pyrimidine Salvage ◽  
Key Enzyme

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The structure of the C212S mutant of uridine phosphorylase from the facultatively aerobic Gram-negative γ-proteobacteriumShewanella oneidensisMR-1 (SoUP) was determined at 1.68 Å resolution. A comparison of the structures of the mutant and the wild-type enzyme showed that one dimer in the mutant hexamer differs from all other dimers in the mutant and wild-type SoUP (both in the free form and in complex with uridine). The key difference is the `maximum open' state of one of the subunits comprising this dimer, which has not been observed previously for uridine phosphorylases. Some conformational features of the SoUP dimer that provide access of the substrate into the active site are revealed. The binding of the substrate was shown to require the concerted action of two subunits of the dimer. The changes in the three-dimensional structure induced by the C212S mutation account for the lower affinity of the mutant for inorganic phosphate, while the affinity for uridine remains unchanged.

scholarly journals Deciphering the Role of Residues Involved in Disorder-To-Order Transition Regions in Archaeal tRNA Methyltransferase 5

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 399
Author(s):  
Ambuj Srivastava ◽  
Dhanusha Yesudhas ◽  
Shandar Ahmad ◽  
M. Michael Gromiha
Keyword(s):  
Three Dimensional ◽  
Md Simulations ◽  
Order Transition ◽  
Free Form ◽  
Wild Type ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Type Complex ◽  
In The Wild ◽  
Trna Methyltransferase

tRNA methyltransferase 5 (Trm5) enzyme is an S-adenosyl methionine (AdoMet)-dependent methyltransferase which methylates the G37 nucleotide at the N1 atom of the tRNA. The free form of Trm5 enzyme has three intrinsically disordered regions, which are highly flexible and lack stable three-dimensional structures. These regions gain ordered structures upon the complex formation with tRNA, also called disorder-to-order transition (DOT) regions. In this study, we performed molecular dynamics (MD) simulations of archaeal Trm5 in free and complex forms and observed that the DOT residues are highly flexible in free proteins and become stable in complex structures. The energetic contributions show that DOT residues are important for stabilising the complex. The DOT1 and DOT2 are mainly observed to be important for stabilising the complex, while DOT3 is present near the active site to coordinate the interactions between methyl-donating ligands and G37 nucleotides. In addition, mutational studies on the Trm5 complex showed that the wild type is more stable than the G37A tRNA mutant complex. The loss of productive interactions upon G37A mutation drives the AdoMet ligand away from the 37th nucleotide, and Arg145 in DOT3 plays a crucial role in stabilising the ligand, as well as the G37 nucleotide, in the wild-type complex. Further, the overall energetic contribution calculated using MMPBSA corroborates that the wild-type complex has a better affinity between Trm5 and tRNA. Overall, our study reveals that targeting DOT regions for binding could improve the inhibition of Trm5.

scholarly journals Structural analysis of mutations in the Drosophila beta 2-tubulin isoform reveals regions in the beta-tubulin molecular required for general and for tissue-specific microtubule functions.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 267-286 ◽  
Author(s):  
J D Fackenthal ◽  
J A Hutchens ◽  
F R Turner ◽  
E C Raff
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Variable Region ◽  
Internal Variable ◽  
Microtubule Assembly ◽  
Dimensional Structure ◽  
Wild Type ◽  
Beta Tubulin ◽  
Assembly Kinetics ◽  
Beta 2

Abstract We have determined the lesions in a number of mutant alleles of beta Tub85D, the gene that encodes the testis-specific beta 2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the beta 2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all beta-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t6 contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate beta-tubulins. Correspondingly, B2t6 disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that beta 3, a developmentally regulated Drosophila beta-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type beta 2-tubulin. We show here by complementation analysis that beta 3 and the B2t6 product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the beta 2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the beta 2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the beta 2 variant lacking the carboxy terminus and the B2t6 variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate beta-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type beta-tubulins. We propose that the integrity of this structure in the Drosophila testis beta 2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.

scholarly journals Solvent Accessibility of Residues Undergoing Pathogenic Variations in Humans: From Protein Structures to Protein Sequences

2021 ◽  
Vol 7 ◽  
Author(s):  
Castrense Savojardo ◽  
Matteo Manfredi ◽  
Pier Luigi Martelli ◽  
Rita Casadio
Keyword(s):  
Solvent Accessibility ◽  
Protein Structures ◽  
Three Dimensional ◽  
Protein Sequences ◽  
Large Data ◽  
Human Protein ◽  
Dimensional Structure ◽  
Wild Type ◽  
Solvent Exposure ◽  
Data Set

Solvent accessibility (SASA) is a key feature of proteins for determining their folding and stability. SASA is computed from protein structures with different algorithms, and from protein sequences with machine-learning based approaches trained on solved structures. Here we ask the question as to which extent solvent exposure of residues can be associated to the pathogenicity of the variation. By this, SASA of the wild-type residue acquires a role in the context of functional annotation of protein single-residue variations (SRVs). By mapping variations on a curated database of human protein structures, we found that residues targeted by disease related SRVs are less accessible to solvent than residues involved in polymorphisms. The disease association is not evenly distributed among the different residue types: SRVs targeting glycine, tryptophan, tyrosine, and cysteine are more frequently disease associated than others. For all residues, the proportion of disease related SRVs largely increases when the wild-type residue is buried and decreases when it is exposed. The extent of the increase depends on the residue type. With the aid of an in house developed predictor, based on a deep learning procedure and performing at the state-of-the-art, we are able to confirm the above tendency by analyzing a large data set of residues subjected to variations and occurring in some 12,494 human protein sequences still lacking three-dimensional structure (derived from HUMSAVAR). Our data support the notion that surface accessible area is a distinguished property of residues that undergo variation and that pathogenicity is more frequently associated to the buried property than to the exposed one.

scholarly journals Hydrogen deuterium exchange defines catalytically linked regions of protein flexibility in the catechol O-methyltransferase reaction

2020 ◽  
Vol 117 (20) ◽  
pp. 10797-10805
Author(s):  
Jianyu Zhang ◽  
Jeremy L. Balsbaugh ◽  
Shuaihua Gao ◽  
Natalie G. Ahn ◽  
Judith P. Klinman
Keyword(s):  
Mass Spectrometric Study ◽  
Break Point ◽  
Deuterium Exchange ◽  
Primary Group ◽  
Hydrogen Deuterium Exchange ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
In The Wild ◽  
Donor Acceptor ◽  
Hydrogen Deuterium

Human catechol O-methyltransferase (COMT) has emerged as a model for understanding enzyme-catalyzed methyl transfer from S-adenosylmethionine (AdoMet) to small-molecule catecholate acceptors. Mutation of a single residue (tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor–acceptor compaction within the activated ground state of the wild type enzyme [J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U.S.A. 112, 7954–7959 (2015)]. This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15–20 °C indicates changes in flexibility across ∼40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can be understood in the context of a mutationally induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate.

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