A novel alpha-ketoglutarate reductase activity of the serA-encoded 3-phosphoglycerate dehydrogenase of Escherichia coli K-12 and its possible implications for human 2-hydroxyglutaric aciduria

A set of asymmetric hybrid tetramers of Escherichia coli D-3-phosphoglycerate dehydrogenase (PGDH) have been made by gene co-expression and KSCN-induced dimer exchange. These tetramers contain varied numbers of active sites and effector binding sites arranged in different orientations within the tetramer. They reveal that PGDH displays half-of-the-sites activity with respect to its active sites and that the two sites that are active at any particular time lie in subunits on either side of the nucleotide binding domain interface. Half-ofthe-sites functionality is also observed for the effector even though all four sites eventually bind effector. That is, only two effector sites need to be occupied for maximum inhibition. Binding of the last two effector molecules does not contribute functionally to inhibition of activity. Furthermore, positive cooperativity of inhibition of activity by the effector is completely dependent on the positive cooperativity of binding of the effector. Binding of the first effector molecule produces a conformational change that essentially completely inhibits the active site within the subunit to which it binds and produces an approximate 33% inhibition of the active site in the subunit to which it is not bound. Binding of the second effector at the opposite regulatory domain interface completes the inhibition of activity. This simple relationship defines the positional and quantitative influence of effector ligand binding on activity and can be used to predict the maximum level of inhibition of individual hybrid tetramers. In addition, the site-specific quantitative relationship of effector binding to individual active sites can be used to model the inhibition profile with relatively good agreement. These simple rules for the site to site interaction in PGDH provide significant new insight into the mechanism of allostery of this enzyme.D-3-Phosphoglycerate dehydrogenase (PGDH) 1 (EC 1.1.1.95) catalyzes the first committed step in the biosynthesis of Lserine (1-2). The enzyme displays normal Michaelis-Menten kinetics, but is inhibited in a cooperative allosteric manner by L-serine with a Hill coefficient of ϳ2 (1-5).PGDH is a tetramer composed of identical subunits that contain three distinct structural domains, the substrate binding domain, the nucleotide binding domain, and the regulatory or effector binding domain (6). The arrangement of subunits reveals that the enzyme can be viewed as a dimer of dimers. Contact between the nucleotide binding domains of 2 subunits form one dimer and contact between the regulatory domains of two of these dimers complete the tetramer contacts (see Fig. 1). L-Serine binds at the interface between adjacent regulatory domains as illustrated in Fig. 2. There are two such interfaces at opposite ends of the tetramer and 2 serine molecules bind in each interface forming hydrogen-bonding contacts across the interface. It has been proposed that the binding of L-serine stabilizes the regulatory domain interface contacts and inhibits the active site by imparting...

“…Enzyme activity was determined by the change in absorbance at 340 nm because of the conversion of NADH to NAD ϩ at pH 7.5 (13) using ␣-ketoglutarate as the substrate (14).…”

Section: Methodsmentioning

confidence: 99%

Quantitative Relationships of Site to Site Interaction in Escherichia coli d-3-Phosphoglycerate Dehydrogenase Revealed by Asymmetric Hybrid Tetramers

Grant

2004

“…Enzyme activity was determined by the change in absorbance at 340 nm due to the conversion of NADH to NAD ϩ at pH 7.5 (18) using ␣-ketoglutarate as the substrate (19).…”

Section: Methodsmentioning

confidence: 99%

Hybrid Tetramers Reveal Elements of Cooperativity in Escherichia colid-3-Phosphoglycerate Dehydrogenase

Grant

2003

D-3-Phosphoglycerate dehydrogenase from Escherichia coli is a tetramer of identical subunits that is inhibited when L-serine binds at allosteric sites between subunits. Co-expression of two genes, the native gene containing a charge difference mutation and a gene containing a mutation that eliminates serine binding, produces hybrid tetramers that can be separated by ion exchange chromatography. Activity in the hybrid tetramer with only a single intact serine binding site is inhibited by ϳ58% with a Hill coefficient of 1. Thus, interaction at a single regulatory domain interface does not, in itself, lead to the positive cooperativity of inhibition manifest in the native enzyme. Tetramers with only two intact serine binding sites purify as a mixture that displays a maximum inhibition level that is less than that of native enzyme, suggesting the presence of a population of tetramers that are unable to be fully inhibited. Differential analysis of this mixture supports the conclusion that it contains two forms of the tetramer. One form contains two intact serine binding sites at the same interface and is not fully inhibitable. The second form is a fully inhibitable population that has one serine binding site at each interface. Overall, the hybrid tetramers show that the positive cooperativity observed for serine binding is mediated across the nucleotide binding domain interface, and the negative cooperativity is mediated across the regulatory domain interface. That is, they reveal a pattern in which the binding of serine at one interface leads to negative cooperativity of binding of a subsequent serine at the same interface and positive cooperativity of binding of a subsequent serine to the opposite interface. This trend is propagated to subsequent binding sites in the tetramer such that the negative cooperativity that is originally manifest at one interface is decreased by subsequent binding of ligand at the opposite interface.

“…It has previously been shown that E. coli as well as human phosphoglycerate dehydrogenase can reduce ␣-ketoglutarate (a close structural analog of 3-phosphohydroxypyruvate) to D-2HG in addition to catalyzing their main physiological reaction, which consists of the conversion of 3-phosphoglycerate to 3-phosphohydroxypyruvate in the first step of serine biosynthesis (14,49). Furthermore, in S. cerevisiae, the gene expression profiles of DLD3 and SER33, one of the two yeast phosphoglycerate dehydrogenase isoforms, are highly correlated across several datasets (SPELL version 2.0.3 (50)).…”

Section: Effect Of Modulating Intracellular Yeast Phosphoglycerate Dementioning

confidence: 99%

Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase

Becker-Kettern

Paczia

Conrotte

et al. 2016

122

The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high D-2-hydroxyglutarate (D-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3⌬ knock-out strain accumulates millimolar levels of D-2HG, whereas a dld2⌬ knock-out strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to ␣-ketoglutarate. Depletion of D-lactate levels in the dld3⌬, but not in the dld2⌬ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to ␣-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce ␣-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1. 2-Hydroxyglutarate (2HG)3 is a 5-carbon dicarboxylic acid that was first detected in human urine in the late 1970s (1).Because of the hydroxyl group on the second carbon, 2HG exists under two enantiomeric configurations (L or D) that can be separated by gas or liquid chromatography after derivatization with another chiral compound and that can therefore be differentially assayed in biological samples using GC-MS or LC-MS methods (2, 3). The interest in 2HG increased when it was found to accumulate in urine of patients with suspected inborn errors of metabolism (4, 5). Most 2-hydroxyglutaric aciduria patients present elevations of either L-2HG or D-2HG in their extracellular fluids, and the clinical phenotype depends on the configuration of the accumulated organic acid. More recently, cases of "combined D,L-hydroxyglutaric aciduria" have been reported (6).2-Hydroxyglutaric acidurias remained enigmatic diseases because neither L-2HG nor D-2HG are intermediates of any known metabolic pathway, and the causal gene deficiencies were only discovered many years after the first patient case reports. It is now established that L-2-hydroxyglutaric aciduria is caused by loss-of-function mutations in the L2HGDH gene, encoding a specific L-2HG dehydrogenase, whereas in many cases D-2-hydroxyglutaric aciduria results from loss-of-function mutations in the D2H...