Enzymatic Characterization (enzymatic + characterization)

Distribution by Scientific Domains


Selected Abstracts


Mouse cytosolic sulfotransferase SULT2B1b interacts with cytoskeletal proteins via a proline/serine-rich C-terminus

FEBS JOURNAL, Issue 18 2010
Katsuhisa Kurogi
Cytosolic sulfotransferase (SULT) SULT2B1b had previously been characterized as a cholesterol sulfotransferase. Like human SULT2B1, mouse SULT2B1b contains a unique, 31 amino acid C-terminal sequence with a proline/serine-rich region, which is not found in members of other SULT families. To gain insight into the functional relevance of this proline/serine-rich region, we constructed a truncated mouse SULT2B1b lacking the 31 C-terminal amino acids, and compared it with the wild-type enzyme. Enzymatic characterization indicated that the catalytic activity was not significantly affected by the absence of those C-terminal residues. Glutathione S -transferase pulldown assays showed that several proteins interacted with mouse SULT2B1b specifically through this C-terminal proline/serine-rich region. Peptide mass fingerprinting revealed that of the five SULT2B1b-binding proteins analyzed, three were cytoskeletal proteins and two were cytoskeleton-binding molecular chaperones. Furthermore, wild-type mouse SULT2B1b, but not the truncated enzyme, was associated with the cytoskeleton in experiments with a cytoskeleton-stabilizing buffer. Collectively, these results suggested that the unique, extended proline/serine-rich C-terminus of mouse SULT2B1b is important for its interaction with cytoskeletal proteins. Such an interaction may allow the enzyme to move along microfilaments such as actin filaments, and catalyze the sulfation of hydroxysteroids, such as cholesterol and pregnenolone, at specific intracellular locations. Structured digital abstract ,,MINT-7975854: Sult2B1b (uniprotkb:O35400) physically interacts (MI:0914) with Myosin-Ic (uniprotkb:Q9WTI7), Alpha-actinin-1 (uniprotkb:Q7TPR4), Alpha-actinin-4 (uniprotkb:P57780), HSP 90-beta (uniprotkb:P11499), Hsc70, (uniprotkb:P63017), Beta-actin (uniprotkb:P60710) and Gamma-actin (uniprotkb:P63260) by pull down (MI:0096) [source]


Biochemical characterization of rice trehalose-6-phosphate phosphatases supports distinctive functions of these plant enzymes

FEBS JOURNAL, Issue 5 2007
Shuhei Shima
Substantial levels of trehalose accumulate in bacteria, fungi, and invertebrates, where it serves as a storage carbohydrate or as a protectant against environmental stresses. In higher plants, trehalose is detected at fairly low levels; therefore, a regulatory or signaling function has been proposed for this molecule. In many organisms, trehalose-6-phosphate phosphatase is the enzyme governing the final step of trehalose biosynthesis. Here we report that OsTPP1 and OsTPP2 are the two major trehalose-6-phosphate phosphatase genes expressed in vegetative tissues of rice. Similar to results obtained from our previous OsTPP1 study, complementation analysis of a yeast trehalose-6-phosphate phosphatase mutant and activity measurement of the recombinant protein demonstrated that OsTPP2 encodes a functional trehalose-6-phosphate phosphatase enzyme. OsTPP2 expression is transiently induced in response to chilling and other abiotic stresses. Enzymatic characterization of recombinant OsTPP1 and OsTPP2 revealed stringent substrate specificity for trehalose 6-phosphate and about 10 times lower Km values for trehalose 6-phosphate as compared with trehalose-6-phosphate phosphatase enzymes from microorganisms. OsTPP1 and OsTPP2 also clearly contrasted with microbial enzymes, in that they are generally unstable, almost completely losing activity when subjected to heat treatment at 50 C for 4 min. These characteristics of rice trehalose-6-phosphate phosphatase enzymes are consistent with very low cellular substrate concentration and tightly regulated gene expression. These data also support a plant-specific function of trehalose biosynthesis in response to environmental stresses. [source]


Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysis

PROTEIN SCIENCE, Issue 6 2008
Dalei Wu
Abstract Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L-alanine and D-alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D-cycloserine-bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D- to L-Ala and L- to D-Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr. [source]


The allene oxide cyclase family of Arabidopsis thaliana , localization and cyclization

FEBS JOURNAL, Issue 10 2008
Florian Schaller
Jasmonates are derived from oxygenated fatty acids (oxylipins) via the octadecanoid pathway and are characterized by a pentacyclic ring structure. They have regulatory functions as signaling molecules in plant development and adaptation to environmental stress. Recently, we solved the structure of allene oxide cyclase 2 (AOC2) of Arabidopsis thaliana, which is, together with the other three AOCs, a key enzyme in the biosynthesis of jasmonates, in that it releases the first cyclic and biologically active metabolite , 12-oxo-phytodienoic acid (OPDA). On the basis of models for the bound substrate, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, and the product, OPDA, we proposed that a conserved Glu promotes the reaction by anchimeric assistance. According to this hypothesis, the transition state with a pentadienyl carbocation and an oxyanion is stabilized by a strongly bound water molecule and favorable ,,, interactions with aromatic residues in the cavity. Stereoselectivity results from steric restrictions to the necessary substrate isomerizations imposed by the protein environment. Here, site-directed mutagenesis was used to explore and verify the proposed reaction mechanism. In a comparative analysis of the AOC family from A. thaliana involving enzymatic characterization, in vitro import, and transient expression of AOC,enhanced green fluorescent protein fusion proteins for analysis of subcellular targeting, we demonstrate that all four AOC isoenzymes may contribute to jasmonate biosynthesis, as they are all located in chloroplasts and, in concert with the allene oxide synthase, they are all able to convert 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid into enantiomerically pure cis(+)-OPDA. [source]


The serine palmitoyltransferase from Sphingomonas wittichii RW1: An interesting link to an unusual acyl carrier protein

BIOPOLYMERS, Issue 9 2010
Marine C. C. Raman
Abstract Serine palmitoyltransferase (SPT) catalyses the first step in the de novo biosynthesis of sphingolipids (SLs). It uses a decarboxylative Claisen-like condensation reaction to couple L -serine with palmitoyl-CoA to generate a long-chain base product, 3-ketodihydrosphingosine. SLs are produced by mammals, plants, yeast, and some bacteria, and we have exploited the complete genome sequence of Sphingomonas wittichii to begin a complete analysis of bacterial sphingolipid biosynthesis. Here, we describe the enzymatic characterization of the SPT from this organism and present its high-resolution x-ray structure. Moreover, we identified an open reading frame with high sequence homology to acyl carrier proteins (ACPs) that are common to fatty acid biosynthetic pathways. This small protein was co-expressed with the SPT and we isolated and characterised the apo- and holo-forms of the ACP. Our studies suggest a link between fatty acid and sphingolipid metabolism. 2010 Wiley Periodicals, Inc. Biopolymers 93: 811,822, 2010. [source]