Arylamine N-acetyltransferases: a pharmacogenomic approach to drug metabolism and endogenous function

2003 ◽  
Vol 31 (3) ◽  
pp. 615-619 ◽  
Author(s):  
E. Sim ◽  
K. Pinter ◽  
A. Mushtaq ◽  
A. Upton ◽  
J. Sandy ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Three Dimensional ◽  
Catalytic Triad ◽  
Cysteine Residue ◽  
Structural Studies ◽  
Active Site Cysteine ◽  
Polymorphic Genes ◽  
Genetic Modifications ◽  
Acetyl Group ◽  
Hypertensive Drug

The arylamine N-acetyltransferases (NATs) are a unique family of enzymes that catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of hydrazine and arylamine drugs and carcinogens. The NATs have been shown to be important in drug detoxification and carcinogen activation, with humans possessing two isoenzymes encoded by polymorphic genes. This polymorphism has pharmacogenetic implications, leading to different rates of inactivation of drugs, including the anti-tubercular agent isoniazid and the anti-hypertensive drug hydralazine. Mice provide a good model for human NAT, allowing genetic manipulation of expression to explore possible endogenous roles of these enzymes. The first three-dimensional NAT structure was resolved for NAT from Salmonella typhimurium, and subsequently the structure of NAT from Mycobacterium smegmatis has been elucidated. These identified a ‘Cys-His-Asp’ catalytic triad (conserved in all NATs), which is believed to be responsible for the activation of the active site cysteine residue. As more genomic data become available, NAT homologues continue to be found in prokaryotic species, many of which are pathogenic, including Mycobacterium tuberculosis. The discovery of NAT in M. tuberculosis is particularly significant, since this enzyme participates in inactivation of isoniazid in the bacterium, with implications for isoniazid resistance. Structural studies on NAT proteins and phenotypic analyses of organisms (both mice and prokaryotes) following genetic modifications of the nat genes are leading to an understanding of the potentially diverse roles of NAT in endogenous and xenobiotic metabolism. These studies have indicated that NAT, particularly in Mycobacteria, has the potential to be a drug target. Combinatorial chemical approaches, together with in silico structural studies, will allow for advances in the identification of NAT substrates and inhibitors, both as experimental tools and as potential drugs.

Architecture of the catalytic HPN motif is conserved in all E2 conjugating enzymes

2012 ◽  
Vol 445 (2) ◽  
pp. 167-174 ◽  
Author(s):  
Benjamin W. Cook ◽  
Gary S. Shaw
Keyword(s):  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Imidazole Ring ◽  
Cysteine Residue ◽  
Amide Proton ◽  
Active Site Cysteine ◽  
Shift Analysis ◽  
Conjugating Enzymes ◽  
E2 Enzymes

E2 conjugating enzymes are the central enzymes in the ubiquitination pathway and are responsible for the transfer of ubiquitin and ubiquitin-like proteins on to target substrates. The secondary structural elements of the catalytic domain of these enzymes is highly conserved, including the sequence conservation of a three-residue HPN (His–Pro–Asn) motif located upstream of the active-site cysteine residue used for ubiquitin conjugation. Despite the vast structural knowledge of E2 enzymes, the catalytic mechanism of these enzymes remains poorly understood, in large part due to variation in the arrangements of the residues in the HPN motif in existing E2 structures. In the present study, we used the E2 enzyme HIP2 to probe the structures of the HPN motif in several other E2 enzymes. A combination of chemical-shift analysis, determination of the histidine protonation states and amide temperature coefficients were used to determine the orientation of the histidine ring and hydrogen-bonding arrangements within the HPN motif. Unlike many three-dimensional structures, we found that a conserved hydrogen bond between the histidine imidazole ring and the asparagine backbone amide proton, a common histidine protonation state, and a common histidine orientation exists for all E2 enzymes examined. These results indicate that the histidine within the HPN motif is orientated to structurally stabilize a tight turn motif in all E2 enzymes and is not orientated to interact with the asparagine side chain as proposed in some mechanisms. These results suggest that a common catalysis mechanism probably exists for all E2 conjugating enzymes to facilitate ubiquitin transfer.

Ribosome Structural Organization

1974 ◽  
Vol 32 ◽  
pp. 332-333
Author(s):  
James A. Lake
Keyword(s):  
Ribosomal Proteins ◽  
Structural Organization ◽  
Three Dimensional ◽  
Small Subunit ◽  
Structural Studies ◽  
X Ray Diffraction ◽  
Specific Antibodies ◽  
X Ray ◽  
E Coli ◽  
Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

Three-Dimensional Structure of Rotavirus-Fab Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 238-239
Author(s):  
B.V.V. Prasad ◽  
E. Marietta ◽  
J.W. Burns ◽  
M.K. Estes ◽  
W. Chiu
Keyword(s):  
Image Processing ◽  
Smooth Surface ◽  
Three Dimensional ◽  
Outer Shell ◽  
Dimensional Structure ◽  
Structural Studies ◽  
Cell Penetration ◽  
Electron Cryomicroscopy ◽  
Image Processing Techniques ◽  
Processing Techniques

Rotaviruses are spherical, double-shelled particles. They have been identified as a major cause of infantile gastroenteritis worldwide. In our earlier studies we determined the three-dimensional structures of double-and single-shelled simian rotavirus embedded in vitreous ice using electron cryomicroscopy and image processing techniques to a resolution of 40Å. A distinctive feature of the rotavirus structure is the presence of 132 large channels spanning across both the shells at all 5- and 6-coordinated positions of a T=13ℓ icosahedral lattice. The outer shell has 60 spikes emanating from its relatively smooth surface. The inner shell, in contrast, exhibits a bristly surface made of 260 morphological units at all local and strict 3-fold axes (Fig.l).The outer shell of rotavirus is made up of two proteins, VP4 and VP7. VP7, a glycoprotein and a neutralization antigen, is the major component. VP4 has been implicated in several important functions such as cell penetration, hemagglutination, neutralization and virulence. From our earlier studies we had proposed that the spikes correspond to VP4 and the rest of the surface is composed of VP7. Our recent structural studies, using the same techniques, with monoclonal antibodies specific to VP4 have established that surface spikes are made up of VP4.

scholarly journals Structure-Based Drug Design and Characterization of Sulfonyl-Piperazine Benzothiazinone Inhibitors of DprE1 from Mycobacterium tuberculosis

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Jérémie Piton ◽  
Anthony Vocat ◽  
Andréanne Lupien ◽  
Caroline S. Foo ◽  
Olga Riabova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Design ◽  
Antitubercular Activity ◽  
Cysteine Residue ◽  
Drug Candidate ◽  
Structure Based Drug Design ◽  
Content Type ◽  
Active Site Cysteine ◽  
Covalent Adducts

ABSTRACT Macozinone (MCZ) is a tuberculosis (TB) drug candidate that specifically targets the essential flavoenzyme DprE1, thereby blocking synthesis of the cell wall precursor decaprenyl phosphoarabinose (DPA) and provoking lysis of Mycobacterium tuberculosis. As part of the MCZ backup program, we exploited structure-guided drug design to produce a new series of sulfone-containing derivatives, 2-sulfonylpiperazin 8-nitro 6-trifluoromethyl 1,3-benzothiazin-4-one, or sPBTZ. These compounds are less active than MCZ but have a better solubility profile, and some derivatives display enhanced stability in microsomal assays. DprE1 was efficiently inhibited by sPBTZ, and covalent adducts with the active-site cysteine residue (C387) were formed. However, despite the H-bonding potential of the sulfone group, no additional bonds were seen in the crystal structure of the sPBTZ-DprE1 complex with compound 11326127 compared to MCZ. Compound 11626091, the most advanced sPBTZ, displayed good antitubercular activity in the murine model of chronic TB but was less effective than MCZ. Nonetheless, further testing of this MCZ backup compound is warranted as part of combination treatment with other TB drugs.

scholarly journals The location of the active-site histidine residue in the primary sequence of papain

1968 ◽  
Vol 108 (5) ◽  
pp. 861-866 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Amino Acid ◽  
Sodium Borohydride ◽  
Active Site ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Primary Sequence ◽  
Active Site Cysteine ◽  
Active Site Histidine ◽  
Active Site Cysteine Residue

Papain that had been irreversibly inhibited with 1,3-dibromo[2−14C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin the radioactive peptides were purified chromatographically. Their amino acid composition indicated that cysteine-25 and histidine-106 were cross-linked. Since cysteine-25 is known to be the active-site cysteine residue, histidine-106 must be the active-site histidine residue.

Abstract 408: Essential Domain Transition Involving Central Helical Repeats Prevent Lipid-Free ApoA-I from Acquiring Lipid

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Ricquita Pollard ◽  
Brian Fulp ◽  
Michael Thomas ◽  
Mary Sorci-Thomas
Keyword(s):  
Three Dimensional ◽  
Density Lipoprotein ◽  
Particle Formation ◽  
Cysteine Residue ◽  
Lipid Binding ◽  
Structural Topology ◽  
Loss Of Function ◽  
Mobility Shift ◽  
Helix Bundle ◽  
Plasma High Density

Plasma high density lipoprotein (HDL) concentration is negatively correlated with the occurrence of coronary heart disease in the human population. Because apoA-I is the main protein constituent of HDL, a thorough understanding of apoA-I structural topology is essential for elucidating its ability to package and mobilize cholesterol for catabolism. To determine which of the 10 helical repeats within apoA-I participates in the structural transitions that drive unfolding of the 4-helix bundle, we created several loss of function mutations. Published three-dimensional coordinates of full-length lipid-free apoA-I were used to predict amino acids having spatial separations of 3-5Å within the 4-helix bundle. Based on these predictions, we proposed that specific targeted double cysteine residue substitutions could form disulfide linkages and prevent “opening” of a critical domain required for unfolding of the apoA-I 4-helix bundle when exposed to lipid. To test the importance of helical repeats 4, 5, 6 and 7, double cysteine mutants D103C-R177C apoA-I and F104C-H162C apoA-I were created, expressed and purified using established procedures. Mass spectrometry combined with MS/MS sequencing was used to verify the “locked” disulfide form of each double cysteine substitution mutants. Using 20% SDS-PAGE we show that electrophoretic mobility-shift distinguishes between “locked” or -DTT versus “unlocked” or +DTT form for each of the mutant apoA-I proteins. Particle formation was tested for each mutant by measuring the formation of recombinant HDL (rHDL) using cholate dialysis, as well as, the formation of nascent HDL (nHDL) from ABCA1 expressing cells. Examination of the size of rHDL and nHDL particles formed suggests that unfolding of lipid-free apoA-I to acquire lipid involves the “locked” or restricted helical repeats 4-7. In conclusion, when both double cysteine apoA-I mutants exist in their “locked” conformation evidence of impaired particle formation was observed confirming the existence of the apoA-I 4 helix bundle in lipid free state and the role of central helical repeats 4-7 in lipid binding and particle formation.

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