Substrate recognition sites in 14α-sterol demethylase from comparative analysis of amino acid sequences and X-ray structure of Mycobacterium tuberculosis CYP51

2001 ◽  
Vol 87 (4) ◽  
pp. 227-235 ◽  
Author(s):  
Larissa M Podust ◽  
Jure Stojan ◽  
Thomas L Poulos ◽  
Michael R Waterman
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Comparative Analysis ◽  
Amino Acid ◽  
Substrate Recognition ◽  
Amino Acid Sequences ◽  
X Ray ◽  
Recognition Sites

Resolution of a protein sequence ambiguity by X-ray crystallographic and mass spectrometric methods

1992 ◽  
Vol 25 (2) ◽  
pp. 205-210 ◽  
Author(s):  
L. J. Keefe ◽  
E. E. Lattman ◽  
C. Wolkow ◽  
A. Woods ◽  
M. Chevrier ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Electron Density ◽  
Mass Spectrometric ◽  
Amino Acid Sequences ◽  
Data Matrix ◽  
Protein Crystal ◽  
Insertion Mutant ◽  
Laser Desorption Mass Spectrometry ◽  
X Ray

Ambiguities in amino acid sequences are a potential problem in X-ray crystallographic studies of proteins. Amino acid side chains often cannot be reliably identified from the electron density. Many protein crystal structures that are now being solved are simple variants of a known wild-type structure. Thus, cloning artifacts or other untoward events can readily lead to cases in which the proposed sequence is not correct. An example is presented showing that mass spectrometry provides an excellent tool for analyzing suspected errors. The X-ray crystal structure of an insertion mutant of Staphylococcal nuclease has been solved to 1.67 Å resolution and refined to a crystallographic R value of 0.170 [Keefe & Lattman (1992). In preparation]. A single residue has been inserted in the C-terminal α helix. The inserted amino acid was believed to be an alanine residue, but the final electron density maps strongly indicated that a glycine had been inserted instead. To confirm the observations from the X-ray data, matrix-assisted laser desorption mass spectrometry was employed to verify the glycine insertion. This mass spectrometric technique has sufficient mass accuracy to detect the methyl group that distinguishes glycine from alanine and can be extended to the more common situation in which crystallographic measurements suggest a problem with the sequence, but cannot pinpoint its location or nature.

scholarly journals A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in Mycobacterium tuberculosis

2020 ◽  
Vol 295 (19) ◽  
pp. 6252-6262 ◽  
Author(s):  
Wanting Jiao ◽  
Yifei Fan ◽  
Nicola J. Blackmore ◽  
Emily J. Parker
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Isothermal Calorimetry ◽  
Conformational Dynamics ◽  
Chorismate Mutase ◽  
Fine Tuning ◽  
Backbone Dynamics ◽  
Single Amino Acid ◽  
Complex Dynamic ◽  
X Ray

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis, which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branchpoint enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering, and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkably, generating a nonallosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.

scholarly journals Comparative Analysis of the Putative Amino Acid Sequences of Chlamydial Heat Shock Protein 60 and Escherichia coli GroEL.

2000 ◽  
Vol 62 (9) ◽  
pp. 941-945 ◽  
Author(s):  
Yoshitsugu OCHIAI ◽  
Hideto FUKUSHI ◽  
Cai YAN ◽  
Tsuyoshi YAMAGUCHI ◽  
Katsuya HIRAI
Keyword(s):  
Escherichia Coli ◽  
Comparative Analysis ◽  
Amino Acid ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Amino Acid Sequences ◽  
Heat Shock Protein 60

scholarly journals Diversity of radA Genes from Cultured and UnculturedArchaea: Comparative Analysis of Putative RadA Proteins and Their Use as a Phylogenetic Marker

1999 ◽  
Vol 181 (3) ◽  
pp. 907-915 ◽  
Author(s):  
Steven J. Sandler ◽  
Philip Hugenholtz ◽  
Christa Schleper ◽  
Edward F. DeLong ◽  
Norman R. Pace ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Amino Acid ◽  
Tissue Sample ◽  
Reca Protein ◽  
Amino Acid Sequences ◽  
Sponge Tissue ◽  
Amino Acid Deletion ◽  
Archaeal Species ◽  
Rrna Sequences ◽  
16S Rrna Sequences

ABSTRACT Archaea-specific radA primers were used with PCR to amplify fragments of radA genes from 11 cultivated archaeal species and one marine sponge tissue sample that contained essentially an archaeal monoculture. The amino acid sequences encoded by the PCR fragments, three RadA protein sequences previously published (21), and two new complete RadA sequences were aligned with representative bacterial RecA proteins and eucaryal Rad51 and Dmc1 proteins. The alignment supported the existence of four insertions and one deletion in the archaeal and eucaryal sequences relative to the bacterial sequences. The sizes of three of the insertions were found to have taxonomic and phylogenetic significance. Comparative analysis of the RadA sequences, omitting amino acids in the insertions and deletions, shows a cladal distribution of species which mimics to a large extent that obtained by a similar analysis of archaeal 16S rRNA sequences. The PCR technique also was used to amplify fragments of 15 radA genes from uncultured natural sources. Phylogenetic analysis of the amino acid sequences encoded by these fragments reveals several clades with affinity, sometimes only distant, to the putative RadA proteins of several species ofCrenarcheota. The two most deeply branching archaealradA genes found had some amino acid deletion and insertion patterns characteristic of bacterial recA genes. Possible explanations are discussed. Finally, signature codons are presented to distinguish among RecA protein family members.

Synopses from the poster exhibition organized by I. M. Kerr, 1. Interferon: a tertiary structure predicted from amino acid sequences

1982 ◽  
Vol 299 (1094) ◽  
pp. 125-127 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
X Ray ◽  
Three Dimensional Structure ◽  
X Ray Crystallography ◽  
Functional Studies

Functional studies on interferon would be helped by a three-dimensional structure for the molecule. However, it may be several years before the structure of the protein is determined by X-ray crystallography. We have therefore used available methods for predicting the secondary - and the tertiary - structure of a protein from its amino acid sequence to propose a tertiary model involving the packing of four a-helices. Details of this work have been published elsewhere (Sternberg & Cohen 1982).

scholarly journals Structural studies on Desulfovibrio gigas cytochrome c3 by two-dimensional 1H-nuclear-magnetic-resonance spectroscopy

1993 ◽  
Vol 294 (3) ◽  
pp. 909-915 ◽  
Author(s):  
M A Piçarra-Pereira ◽  
D L Turner ◽  
J LeGall ◽  
A V Xavier
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Reduced Form ◽  
Desulfovibrio Vulgaris ◽  
Resonance Spectroscopy ◽  
Two Dimensional ◽  
Redox Potentials ◽  
Cytochrome C3 ◽  
X Ray ◽  
Desulfovibrio Gigas

Several aromatic amino acid residues and haem resonances in the fully reduced form of Desulfovibrio gigas cytochrome c3 are assigned, using two-dimensional 1H n.m.r., on the basis of the interactions between the protons of the aromatic amino acids and the haem protons as well as the intrahaem distances known from the X-ray structure [Kissinger (1989) Ph.D. Thesis, Washington State University]. The interhaem interactions observed in the n.m.r. spectra are in full agreement with the D. gigas X-ray structure and also with the n.m.r. data from Desulfovibrio vulgaris (Hildenborough) [Turner, Salgueiro, LeGall and Xavier (1992) Eur. J. Biochem. 210, 931-936]. The good correlation between the calculated ring-current shifts and the observed chemical shifts strongly supports the present assignments. Observation of the two-dimensional nuclear-Overhauser-enhancement spectra of the protein in the reduced, intermediate and fully oxidized stages led to the ordering of the haems in terms of their midpoint redox potentials and their identification in the X-ray structure. The first haem to oxidize is haem I, followed by haems II, III and IV, numbered according to the Cys ligand positions in the amino acid sequences [Mathews (1985) Prog. Biophys. Mol. Biol. 54, 1-56]. Although the haem core architecture is the same for the different Desulfovibrio cytochromes c3, the order of redox potentials is different.

scholarly journals Antigens of Mycobacterium tuberculosis Expressed during Preclinical Tuberculosis: Serological Immunodominance of Proteins with Repetitive Amino Acid Sequences

2001 ◽  
Vol 69 (6) ◽  
pp. 4185-4191 ◽  
Author(s):  
K. K. Singh ◽  
X. Zhang ◽  
A. S. Patibandla ◽  
P. Chien ◽  
S. Laal
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Tandem Repeats ◽  
Response Regulator ◽  
Amino Acid Sequences ◽  
Expression Library ◽  
Immunodeficiency Virus ◽  
Two Component Signal Transduction ◽  
Aerosol Infection

ABSTRACT Four antigens of Mycobacterium tuberculosisthat are expressed in vivo after aerosol infection but prior to the development of clinical tuberculosis (TB) in rabbits were identified by immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained 5 weeks postinfection. Three of the proteins identified, PirG (Rv3810), polymorphic GC-repetitive sequence (PE-PGRS; Rv3367), and proline-threonine repetitive protein (PTRP) (Rv0538), have multiple tandem repeats of unique amino acid sequences and have characteristics of surface or secreted proteins. The fourth protein, MtrA (Rv3246c), is a response regulator of a putative two-component signal transduction system, mtrA-mtrB, ofM. tuberculosis. All four antigens were recognized by pooled sera from TB patients and not from healthy controls, confirming their in vivo expression during active infection in humans. Three of the antigens (PE-PGRS, PTRP, and MtrA) were also recognized by retrospective preclinical TB sera obtained, prior to the clinical manifestation of TB, from human immunodeficiency virus-TB patients, suggesting that they are potential candidates for devising diagnostic tests for active, preclinical TB.

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