Identification of the gene encoding hydroxyacid-oxoacid transhydrogenase, an enzyme that metabolizes 4-hydroxybutyrate

To identify the sequence of hydroxyacid-oxoacid transhydrogenase (HOT), responsible for the oxidation of 4-hydroxybutyrate in mammalian tissues, we have purified this enzyme from rat liver and obtained partial sequences of proteins coeluting with the enzymatic activity in the last purification step. One of the identified proteins was 'iron-dependent alcohol dehydrogenase', an enzyme encoded by a gene present on human chromosome 8q 13.1 and distantly related to bacterial 4-hydroxybutyrate dehydrogenases. The identification of this protein as HOT was confirmed by showing that overexpression of the mouse homologue in HEK cells resulted in the appearance of an enzyme catalyzing the alpha-ketoglutarate-dependent oxidation of 4-hydroxybutyrate to succinate semialdehyde.     

D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum.

The gene encoding D-lactate dehydrogenase (D-lactate: NAD+ oxidoreductase, EC 1.1.1.28) of Lactobacillus plantarum has been sequenced, and expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5'-noncoding region of the gene was replaced with the tac promoter. Comparison of the sequence of D-lactate dehydrogenase with L-lactate dehydrogenases, including the L. plantarum L-lactate dehydrogenase, showed no significant homology. In contrast, the D-lactate dehydrogenase is homologous to E. coli D-3-phosphoglycerate dehydrogenase and Lactobacillus casei D-2-hydroxyisocaproate dehydrogenase. This indicates that D-lactate dehydrogenase is a member of a new family of 2-hydroxyacid dehydrogenases recently proposed, being distinct from L-lactate dehydrogenase and L-malate dehydrogenase, and strongly suggests that the new family consists of D-isomer-stereospecific enzymes. In the reductive reaction, the enzyme showed a broad substrate specificity, although pyruvate was the most favorable of all 2-ketocarboxylic acids tested. In particular, hydroxypyruvate is effectively reduced by the enzyme, the reaction rate, and Km value being comparable to those in the case of pyruvate, indicating that the enzyme has not only D-lactate dehydrogenase activity but also D-glycerate dehydrogenase activity. The conserved residues in this family appear to be the residues involved in the substrate binding and the catalytic reaction, and thus to be targets for site-directed mutagenesis.     

Evolutionary History of <Emphasis Type="SmallCaps">d</Emphasis>-Lactate Dehydrogenases: A Phylogenomic Perspective on Functional Diversity in the FAD Binding Oxidoreductase/Transferase Type 4 Family

Lactate dehydrogenases which convert lactate to pyruvate are found in almost every organism and comprise a group of highly divergent proteins in amino acid sequence, catalytic properties, and substrate specificity. While the l-lactate dehydrogenases are among the most studied enzymes, very little is known about the structure and function of d-lactate dehydrogenases (d-LDHs) which include two discrete classes of enzymes that are classified based on their ability to transfer electrons and/or protons to NAD in NAD-dependent lactate dehydrogenases (nLDHs), and FAD in NAD-independent lactate dehydrogenases (iLDHs). In this study, we used a combination of structural and phylogenomic approaches to reveal the likely evolutionary events in the history of the recently described FAD binding oxidoreductase/transferase type 4 family that led to the evolution of d-iLDHs (commonly referred as DLD). Our phylogenetic reconstructions reveal that DLD genes from eukaryotes form a paraphyletic group with respect to d-2-hydroxyglutarate dehydrogenase (D2HGDH). All phylogenetic reconstructions recovered two divergent yeast DLD phylogroups. While the first group (DLD1) showed close phylogenetic relationships with the animal and plant DLDs, the second yeast group (DLD2) revealed strong phylogenetic and structural similarities to the plant and animal D2HGDH group. Our data strongly suggest that the functional assignment of the yeast DLD2 group should be carefully revisited. The present study demonstrates that structural phylogenomic approach can be used to resolve important evolutionary events in functionally diverse superfamilies and to provide reliable functional predictions to poorly characterized genes.     

A biochemical study of the reconstitution of D-lactate dehydrogenase-deficient membrane vesicles using fluorine-labeled components

Fluorine-19 labeled compounds have been incorporated into lipids and proteins of Escherichia coli. 19F-Labeled membrane vesicles, prepared by growing a fatty acid auxotroph of a D-lactate dehydrogenase-deficient strain on 8,8-difluoromyristic acid, can be reconstituted for oxidase and transport activities by binding exogenous D-lactate dehydrogenase. 19F-Labeled D-lactate dehydrogenases prepared by addition of fluorotryptophans to a tryptophan-requiring strain are able to reconstitute D-lactate dehydrogenase-deficient membrane vesicles. Thus, lipid and protein can be labeled independently and used to investigate protein-lipid interactions in membranes.     

Analysis of ubiquitination in vivo using a transgenic mouse model

The primary pathway for the proteolytic destruction of cellular proteins is through ubiquitin-mediated targeting to the proteasome. This pathway is pivotal not only in the elimination of damaged or misfolded proteins but also in the temporal, developmental, or signal-mediated destruction of normal cellular substrates. The list of known substrates of the ubiquitin/proteasome pathway is long, but most substrates have been identified in yeast or, more recently, in cultured mammalian cells. It is likely that many mammalian substrates with developmental or disease relevance have yet to be identified because their ubiquitination occurs in tissue or organ systems that cannot be adequately modeled in vitro. We have developed a transgenic mouse model that will allow the isolation and identification of these substrates. The human UbC promoter was used to drive expression of a hexahistidine-tagged version of human ubiquitin in a variety of mouse tissues from early embryonic stages, as assessed by a green fluorescent protein marker. Cleavage of the fusion protein by endogenous enzymes produced epitope-tagged ubiquitin that was detected both in monomeric form and conjugated to cellular proteins. This mouse model should facilitate in the analysis of normal and disease-related ubiquitination events in vivo.     

Comparison of the D-lactate stereospecific dehydrogenase of Limulus polyphemus with active-site regions of L-lactate dehydrogenases

Lactate dehydrogenase (D-lactate:NAD+ oxidoreductase, EC 1.1.1.28) from the horseshoe crab, Limulus polyphemus, a dimeric enzyme stereospecific for D-lactate, has been purified by affinity chromatography. Maleyl tryptic peptides containing arginine residues isolated from the Limulus enzyme have been characterized and sequenced. The small peptides obtained from similarly treated L-lactate-specific enzyme homologs define major portions of the substrate and coenzyme binding regions and are virtually identical among L-lactate-specific enzymes. Although the six small peptides and free arginine isolated from the Limulus enzyme indicate that the small number of arginine tryptic peptides are located in a few discrete consecutive clusters similarly to the L-lactate dehydrogenases, the peptides nevertheless show no obvious sequence homology to the corresponding peptides from L-lactate dehydrogenases. These results indicate that this lactate dehydrogenase of altered substrate specificity either evolved with major rearrangements of the active site if it evolved from an L-lactate dehydrogenase, or that D-lactate dehydrogenases have evolved from a different protein. The results contradict proposed models which suggest that minor changes in the spatial orientation of pyruvate resulting from minimal rearrangement of the active site could accommodate the change in substrate specificity.     

Electron acquisition system constructed from an NAD-independent D-lactate dehydrogenase and cytochrome c2 in Rhodopseudomonas palustris No. 7

The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (M(r), about 235,000; subunit M(r) about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50 degrees C, respectively. The Km of the enzyme for D-lactate was 0.8 mM. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxybutyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mM). The enzyme contained a FAD cofactor. Cytochrome c(2) was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c(2) was reduced by incubating with D-LDH and D-lactate.     

Unusual evolution of 11β- and 17β-hydroxysteroid and retinol dehydrogenases

11β-hydroxysteroid dehydrogenases regulate glucocorticoid concentrations and 17β-hydroxysteroid dehydrogenases regulate estrogen and androgen concentrations in mammals. Phylogenetic analysis of the sequences from two 11β-hydroxysteroid dehydrogenases and four mammalian 17β-hydroxysteroid dehydrogenases indicates unusual evolution in these enzymes. Type 1 11β- and 17β-hydroxysteroid dehydrogenases are on the same branch; Type 2 enzymes cluster on another branch with β-hydroxybutyrate dehydrogenase, 11-cis retinol dehydrogenase and retinol dehydrogenase; Type 3 17β-hydroxysteroid dehydrogenase is on a third branch; while the pig dehydrogenase clusters with a yeast multifunctional enzyme on a fourth branch. Pig 17β-hydroxysteroid dehydrogenase appears to have evolved independently from the other three 17β-hydroxysteroid dehydrogenase dehydrogenases; in which case, the evolution of 17β-hydroxysteroid dehydrogenase activity is an example of functional convergence. The phylogeny also suggests that independent evolution of specificity toward C11 substituents on glucocorticoids and C17 substituents on androgens and estrogens has occurred in Types 1 and 2 11β- and 17β-hydroxysteroid dehydrogenases.     

Mass spectrometric identification of human prostate cancer-derived proteins in serum of xenograft-bearing mice

Lack of sensitivity and specificity of current tumor markers has intensified research efforts to find new biomarkers. The identification of potential tumor markers in human body fluids is hampered by large variability and complexity of both control and patient samples, laborious biochemical analyses, and the fact that the identified proteins are unlikely produced by the diseased cells but are due to secondary body defense mechanisms. In a new approach presented here, we eliminate these problems by performing proteomic analysis in a prostate cancer xenograft model in which human prostate cancer cells form a tumor in an immune-incompetent nude mouse. Using this concept, proteins present in mouse serum that can be identified as human will, by definition, originate from the human prostate cancer xenograft and might have potential diagnostic and prognostic value. Using one-dimensional gel electrophoresis, liquid chromatography, and mass spectrometry, we identified tumor-derived human nm23/nucleoside-diphosphate kinase (NME) in the serum of a nude mouse bearing the androgen-independent human prostate cancer xenograft PC339. NME is known to be involved in the metastatic potential of several tumor cells, including prostate cancer cells. Furthermore we identified six human enzymes involved in glycolysis (fructose-bisphosphate aldolase A, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, alpha enolase, and lactate dehydrogenases A and B) in the serum of the tumor-bearing mice. The presence of human NME and glyceraldehyde-3-phosphate dehydrogenase in the serum of PC339-bearing mice was confirmed by Western blotting. Although the putative usefulness of these proteins in predicting prognosis of prostate cancer remains to be determined, the present data illustrate that our approach is a promising tool for the focused discovery of new prostate cancer biomarkers.     

A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells

The tandem affinity purification (TAP) tag technique has been used with success to identify under nondenaturing conditions protein complexes in yeast. The technique can be used in mammalian cells, but we found that the original technique does not yield enough recovery for the identification of proteins when mammalian cells growing in monolayer have to be used. We present here a modified TAP tag technique that allows sufficient recovery of proteins from mouse fibroblasts growing in monolayer cultures. The recovery allows protein identification by mass spectrometry.