D-isomer Specific 2-hydroxyacid Dehydrogenase Research Articles

Illuminating the Structural Determinants for Tetrameric Assembly of Oncogenic CtBP to Guide Inhibitor Design

C-terminal binding proteins (CtBP1 & 2) are co-transcriptional factors that have been implicated in progression of a broad range of cancers. CtBP possesses a D-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domain that binds NAD(H) and provides an attractive target for small molecule intervention. We have previously demonstrated that NAD(H) triggers assembly of CtBP1 and CtBP2 into structurally very similar tetramers, strongly suggesting that this tetrameric form is the co-transcriptionally active species. Using multi-angle light scattering (MALS) to test various NAD(H) moieties, we find that AMP and ADP, but not adenosine, nicotinamide and nicotinamide mononucleotide promote tetramer formation, demonstrating that the adenosine phosphate plays a central role in tetrameric assembly. Crystal structures with bound AMP show a direct structural role of this phosphate in stabilizing a key interaction across the tetrameric interface. Most importantly, these results indicate that development of inhibitors that extend into the nicotinamide pocket, including the regions bound to the nicotinamide ribose and phosphate, should inhibit NAD(H) binding and interfere with tetrameric assembly. We are using these results to guide our development of anti-neoplastic CtBP specific inhibitors.

An aldo-keto reductase with 2-keto-l-gulonate reductase activity functions in l-tartaric acid biosynthesis from vitamin C in Vitis vinifera

Tartaric acid has high economic value as an antioxidant and flavorant in food and wine industries. l-Tartaric acid biosynthesis in wine grape (Vitis vinifera) uses ascorbic acid (vitamin C) as precursor, representing an unusual metabolic fate for ascorbic acid degradation. Reduction of the ascorbate breakdown product 2-keto-l-gulonic acid to l-idonic acid constitutes a critical step in this l-tartaric acid biosynthetic pathway. However, the underlying enzymatic mechanisms remain obscure. Here, we identified a V. vinifera aldo-keto reductase, Vv2KGR, with 2-keto-l-gulonic acid reductase activity. Vv2KGR belongs to the d-isomer–specific 2-hydroxyacid dehydrogenase superfamily and displayed the highest similarity to the hydroxyl pyruvate reductase isoform 2 in Arabidopsis thaliana. Enzymatic analyses revealed that Vv2KGR efficiently reduces 2-keto-l-gulonic acid to l-idonic acid and uses NADPH as preferred coenzyme. Moreover, Vv2KGR exhibited broad substrate specificity toward glyoxylate, pyruvate, and hydroxypyruvate, having the highest catalytic efficiency for glyoxylate. We further determined the X-ray crystal structure of Vv2KGR at 1.58 Å resolution. Comparison of the Vv2KGR structure with those of d-isomer–specific 2-hydroxyacid dehydrogenases from animals and microorganisms revealed several unique structural features of this plant hydroxyl pyruvate reductase. Substrate structural analysis indicated that Vv2KGR uses two modes (A and B) to bind different substrates. 2-Keto-l-gulonic acid displayed the lowest predicted free-energy binding to Vv2KGR among all docked substrates. Hence, we propose that Vv2KGR functions in l-tartaric acid biosynthesis. To the best of our knowledge, this is the first report of a d-isomer–specific 2-hydroxyacid dehydrogenase that reduces 2-keto-l-gulonic acid to l-idonic acid in plants.

Tetrameric Assembly of the Oncogenic C-Terminal Binding Proteins

C-terminal binding proteins (CtBP1 & 2) are co-transcriptional factors that have been implicated in progression of a number of cancers. CtBP possesses a D-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domain that binds NAD(H) and provides an attractive target for small molecule intervention. Our crystal structures of CtBP1 and CtBP2 with bound substrate led to the design of an inhibitor (HIPP) with ∼300nm binding affinity. Experiments using HIPP in APCmin (colon cancer) mouse models suggest that CtBP inhibitors could be efficacious agents in a number of cancers. NAD(H) linked oligomeric assembly of CtBP has been suggested to contribute to co-transcriptional signaling, although the biologically relevant level of CtBP assembly has been unclear. Using multi-angle light scattering (MALS), we find that CtBP assembles from dimers into tetramers as a function of NAD(H) concentration with EC50 values in the range of 50-150 nM, depending on the construct used for CtBP1 and CtBP2. Although crystal structures of CtBP1 and CtBP2 have been described in terms of dimers, due to extensive intradimeric interactions, examination of the distinct crystal lattices for CtBP1 and CtBP2 reveal a very similar tetrameric assembly in both paralogs. Site-directed mutations of residues involved in the observed lattice contacts support the lattice tetramer as the solution tetramer. We propose that this tetramer is the co-transcriptionally active form of CtBP and are investigating inhibitors that disrupt this tetrameric assembly as potential lead compounds for a therapeutically useful CtBP specific inhibitor.

Classification, substrate specificity and structural features of D-2-hydroxyacid dehydrogenases: 2HADH knowledgebase

BackgroundThe family of D-isomer specific 2-hydroxyacid dehydrogenases (2HADHs) contains a wide range of oxidoreductases with various metabolic roles as well as biotechnological applications. Despite a vast amount of biochemical and structural data for various representatives of the family, the long and complex evolution and broad sequence diversity hinder functional annotations for uncharacterized members.ResultsWe report an in-depth phylogenetic analysis, followed by mapping of available biochemical and structural data on the reconstructed phylogenetic tree. The analysis suggests that some subfamilies comprising enzymes with similar yet broad substrate specificity profiles diverged early in the evolution of 2HADHs. Based on the phylogenetic tree, we present a revised classification of the family that comprises 22 subfamilies, including 13 new subfamilies not studied biochemically. We summarize characteristics of the nine biochemically studied subfamilies by aggregating all available sequence, biochemical, and structural data, providing comprehensive descriptions of the active site, cofactor-binding residues, and potential roles of specific structural regions in substrate recognition. In addition, we concisely present our analysis as an online 2HADH enzymes knowledgebase.ConclusionsThe knowledgebase enables navigation over the 2HADHs classification, search through collected data, and functional predictions of uncharacterized 2HADHs. Future characterization of the new subfamilies may result in discoveries of enzymes with novel metabolic roles and with properties beneficial for biotechnological applications.

Computational design and molecular mechanism in oligomerization of C‐terminal binding protein 2

IntroductionC‐terminal binding protein 2 (CtBP2) is moonlighting proteins, which has multiple functions and plays a key role in transcriptional control and in the maintenance of cellular metabolism (1). CtBP2 consist of D‐isomer specific 2‐hydroxyacid dehydrogenase (D2‐HDH) domains and C‐terminal intrinsically unstructured region. Recent studies have shown that the recruitment of a corepressor complex by CtBP2 depends on NADH/NAD ratio. Molecular mechanism of oligomerization of CtBP2 may provide a basis for the formation of a transcriptional corepressor complex.MethodsWe attempted to construct artificial designed proteins on the basis of in silico screening using the INTMSAlign program (2). Overexpression in E. coli cells, and purification of artificial designed CtBP2 (ArCtBP2) were performed. The oligomeric state and the binding affinity to NADH of ArCtBP2 were compared to human CtBP2 (hCtBP2) by size exclusion chromatography and intrinsic Trp fluorescence. To evaluate structural information, X‐ray crystallographic analysis of ArCtBP2 was performed.ResultsSize exclusion chromatography of ArCtBP2 shows 4 peaks for each corresponding oligomeric state, whereas that of hCtBP2 was observed as a single peak. The dissociation constants (Kd) value of NADH to ArCtBP2 was determined as 3.6 ± 0.9 μM by fluorescence spectroscopy. In addition, crystal structure of apo and NAD–bound form of ArCtBP2 at 2.0 and 1.7 Å resolution, respectively. Crystal structure of ArCtBP2 showed opened‐like structure in D2‐HDH domain compared to hCtBP2. These results suggested that the open–closed form of D2‐HDH domain is involved in the binding affinity for NADH and oligomeric state of CtBP2.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 3204: Tryptophan 318/324, the target of C-terminal binding protein (CtBP) inhibitors, plays a critical role in CtBP enzymatic activity, oligomerization and transcriptional coregulation

Abstract C-terminal Binding Proteins (CtBP) 1 and 2 constitute a family of oncogenic transcriptional co-regulators overexpressed in tumor tissues that are associated with worse prognostic outcome and aggressive tumor characteristics in multiple cancer types. Specifically, CtBP has been found to repress expression of genes responsible for apoptosis and EMT (eg. BIK and CDH1) and promote expression of genes that partake in the migration of cancer cells and those that are responsible for enhanced drug resistance (eg. TIAM1 and MDR1). CtBP2 is also critically required for colon cancer stem cell self-renewal. CtBP is unique among transcription co-regulators in harboring a conserved D-isomer specific 2-hydroxyacid dehydrogenase (D2DH) domain, which reduces an alpha-keto acid substrate to an alpha-hydroxy acid in the presence of NADH. The presence of NADH also facilities oligomerization of CtBP, leading to assembly of higher order complexes of CtBP with both DNA binding transcription factors and histone modifying enzymes that then leads to modulation target genes. We have identified hydroxyimino-3-phenylpropanoic acid and its 4-chloro derivative (HIPP; 4-Cl-HIPP) as potent substrate competitive inhibitors of the CtBP dehydrogenase (IC50’s=240nM, 180 nM), which also disrupt CtBP oligomerization, promoter localization, and transcriptional regulation. Co-crystallization of HIPP and CtBP1/2 indicated a strong π-π interaction between the HIPP phenyl ring and the indolyl ring in tryptophan W318/324 of CtBP1/2, indicating that this tryptophan is critical to CtBP interaction with HIPP inhibitors, dehydrogenase function, and quaternary structure. Of note W318/324, though conserved in CtBP1/2, is unique among D2DH, suggesting that a better understanding of W318/324 role and function in CtBP structure and function is critical to optimizing design of inhibitors that targeting this evolutionary unique residue among dehydrogenases. To further elucidate the mechanism of action in catalysis and oligomerization, as well as functional importance for transcription of CtBP2 W324, we analyzed enzyme kinetics, oligomerization, transcriptional co-regulatory activity and cell migration in a series of CtBP2 W324 mutants overexpressed in breast and colon cancer cell lines with concomitant knockdown (siRNA) or knockout (CRISPR/Cas9) of CtBP2. Our data demonstrates that mutation of W324 abrogated dehydrogenase activity, oligomerization, transactivation of the validated CtBP target gene TIAM1 and induction of migration. In summary, our findings suggest that the W324 residue is critical for CtBP2’s function and its unique conservation in CtBP1/2 vs. other dehydrogenases will allow the development of high specificity CtBP W318/324 inhibitors to limit potential toxicity due to off target inhibition of related metabolic dehydrogenases. Citation Format: Martin M. Dcona, Benjamin L. Morris, Priyadarshan K. Damle, Zaid Nawaz, Francisco Zarate Perez, Michael J. Dennis, Sahib J. Singh, William E. Royer, Keith C. Ellis, Steven R. Grossman. Tryptophan 318/324, the target of C-terminal binding protein (CtBP) inhibitors, plays a critical role in CtBP enzymatic activity, oligomerization and transcriptional coregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2017-3204

Structure-guided design of a high affinity inhibitor to human CtBP.

Oncogenic transcriptional coregulators C-terminal Binding Protein (CtBP) 1 and 2 possess regulatory d-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains that provide an attractive target for small molecule intervention. Findings that the CtBP substrate 4-methylthio 2-oxobutyric acid (MTOB) can interfere with CtBP oncogenic activity in cell culture and in mice confirm that such inhibitors could have therapeutic benefit. Recent crystal structures of CtBP 1 and 2 revealed that MTOB binds in an active site containing a dominant tryptophan and a hydrophilic cavity, neither of which are present in other D2-HDH family members. Here, we demonstrate the effectiveness of exploiting these active site features for the design of high affinity inhibitors. Crystal structures of two such compounds, phenylpyruvate (PPy) and 2-hydroxyimino-3-phenylpropanoic acid (HIPP), show binding with favorable ring stacking against the CtBP active site tryptophan and alternate modes of stabilizing the carboxylic acid moiety. Moreover, ITC experiments show that HIPP binds to CtBP with an affinity greater than 1000-fold over that of MTOB, and enzymatic assays confirm that HIPP substantially inhibits CtBP catalysis. These results, thus, provide an important step, and additional insights, for the development of highly selective antineoplastic CtBP inhibitors.

Crystal structures of human CtBP in complex with substrate MTOB reveal active site features useful for inhibitor design

The oncogenic corepressors C-terminal Binding Protein (CtBP) 1 and 2 harbor regulatory d-isomer specific 2-hydroxyacid dehydrogenase (d2-HDH) domains. 4-Methylthio 2-oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD(+) revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD(+) phosphate. Neither feature is present in other d2-HDH enzymes. These structures thus offer key opportunities for the development of highly selective anti-neoplastic CtBP inhibitors.

Genetic evidence that Arabidopsis ALTERED ROOT ARCHITECTURE encodes a putative dehydrogenase involved in homoserine biosynthesis

Genetic and molecular analysis of an Arabidopsis root development mutant identified a putative dehydrogenase gene involved in homoserine biosynthesis. In higher plants, homoserine (Hse) is derived from aspartate (Asp) and is an important intermediate for production of methionine (Met), threonine (Thr), and isoleucine (Ile). In Arabidopsis, six enzymes involved in the biosynthesis of Hse from Asp have been well characterized. It is not known, however, whether there exist other enzymes involved in this process. In this work, we characterized an Arabidopsis mutant, ara (altered root architecture), with a short primary root and an increased number of lateral roots. Genetic and molecular analysis indicated that the ARA gene encodes a protein with a D-isomer specific 2-hydroxyacid dehydrogenase domain. ARA is expressed in all plant organs and is localized in the cell periphery. The ara mutant phenotypes can be rescued by exogenously applied Hse, Met, Ile and 2-oxobutanoate. Based on the results presented here, we propose that the ARA protein may be a dehydrogenase involved in homoserine biosynthesis.

Structural characterization of a D-isomer specific 2-hydroxyacid dehydrogenase from Lactobacillus delbrueckii ssp. bulgaricus

Hydroxyacid dehydrogenases, responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids in lactic acid producing bacteria, have a range of biotechnology applications including antibiotic synthesis, flavor development in dairy products and the production of valuable synthons. The genome of Lactobacillus delbrueckii ssp. bulgaricus, a member of the heterogeneous group of lactic acid bacteria, encodes multiple hydroxyacid dehydrogenases whose structural and functional properties remain poorly characterized. Here, we report the apo and coenzyme NAD⁺ complexed crystal structures of the L. bulgaricusD-isomer specific 2-hydroxyacid dehydrogenase, D2-HDH. Comparison with closely related members of the NAD-dependent dehydrogenase family reveals that whilst the D2-HDH core fold is structurally conserved, the substrate-binding site has a number of non-canonical features that may influence substrate selection and thus dictate the physiological function of the enzyme.

Crystal Structure of d-Erythronate-4-phosphate Dehydrogenase Complexed with NAD

Pyridoxal-5′-phosphate (the active form of vitamin B6) is an essential cofactor in many enzymatic reactions. While animals lack any of the pathways for de novo synthesis and salvage of vitamin B6, it is synthesized by two distinct biosynthetic routes in bacteria, fungi, parasites, and plants. One of them is the PdxA/PdxJ pathway found in the γ subdivision of proteobacteria. It depends on the pdxB gene, which encodes erythronate-4-phosphate dehydrogenase (PdxB), a member of the d-isomer specific 2-hydroxyacid dehydrogenase superfamily. Although three-dimensional structures of other functionally related dehydrogenases are available, no structure of PdxB has been reported. To provide the missing structural information and to gain insights into the catalytic mechanism, we have determined the first crystal structure of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa in the ligand-bound state. It is a homodimeric enzyme consisting of 380-residue subunits. Each subunit consists of three structural domains: the lid domain, the nucleotide-binding domain, and the C-terminal dimerization domain. The latter domain has a unique fold and is largely responsible for dimerization. Interestingly, two subunits of the dimeric enzyme are bound with different combinations of ligands in the crystal and they display significantly different conformations. Subunit A is bound with NAD and a phosphate ion, while subunit B, with a more open active site cleft, is bound with NAD and l(+)-tartrate. Our structural data allow a detailed understanding of cofactor and substrate recognition, thus providing substantial insights into PdxB catalysis.

X-ray structure determination of hydroxyphenylpyruvate reductase at 1.47 Å resolution

Hydroxyphenylpyruvate reductase (HPPR) is involved in the biosynthesis of rosmarinic acid in plants. HPPR was identified, purified and cloned from suspension cultures of Coleus blumei [1] and subsequently expressed from E. coli and purified for crystallization. HPPR belongs to the family of D-isomer specific 2-hydroxyacid dehydrogenases and catalyzes the NAD(P)H dependent reduction of hydroxyphenylpyruvates to the corresponding lactates. HPPR shows only low sequence identity of about 30 % compared to other proteins from this enzyme family. Suitable crystals of HPPR for X-ray diffraction were obtained from 30% MPD, 0.2 M NaCl, pH 7.5 and diffracted to 1.47 A resolution at the Bessy synchrotron. The structure was determined by exhaustive molecular replacement methods. A potential solution obtained with the program PHASER resulted in reasonable electron density for 30% of the molecule. Iterative cycles of automated model building with ARP/wARP resulted in a virtually complete model. The obtained protein structure shows a high structural similarity to other oxidoreductases.

Analysis of 45 kb of DNA located at the left end of the chlorella virus PBCV-1 genorne

Forty-five kilobases of DNA, including the previously sequenced 2.2-kb inverted repeat region, located at the left terminiof the 330-kb Chlorella virus PBCV-1 genome were sequenced and analyzed. Eighty-five complete open reading frames (ORFs) larger than 195 nucleotides were identified. Thirty-seven of the 85 ORFs, which are densely packed on both strands of the DNA, were considered major ORFs. Fifteen of the major ORFs have similarity to genes in the databases, including bacterial glycerophosphoryl diester phosphodiesterase, bacteriophage T4 endonuclease V, d-isomer specific 2-hydroxyacid dehydrogenases, and β-alanine synthetase and bacterial nitrilases. Two major ORFs resemble the virus major capsid protein. Three major ORFs contain three or more ankyrin-like repeat elements and four ORFs encode proline-rich proteins.

Characterization of a nopaline-induced gene for a 40 kDa protein in the nopaline catabolic (noc) region of Ti plasmid pTiC58

The nopaline catabolic region (noc) in Ti plasmid pTiC58 codes for functions in the utilization of nopaline. The enzymes are identified, except for a 40 kDa protein ( 40k gene). The sequence reveals a polypeptide with no homology to other known proteins, except for similarities in two out of three motifs characteristic for d-isomer specific 2-hydroxyacid dehydrogenases.