Determinants of substrate specificity in D-3-phosphoglycerate dehydrogenase. Conversion of the M. tuberculosis enzyme from one that does not use α-ketoglutarate as a substrate to one that does.

d-3-Phosphoglycerate dehydrogenase (PGDH) converts d-3-phosphoglycerate (PGA) to phosphohydroxypyruvate (PHP) in the first step of l-serine biosynthesis. This reaction is reversible, and some PGDHs are capable of using α-ketoglutarate (αKG) instead of PHP in the reverse direction to produce α-hydroxyglutarate. The enzymes so far shown to have this ability are Type II PGDHs, suggesting that this may be a common feature of the Type II enzymes.

D-3-Phosphoglycerate Dehydrogenase.

l-Serine is the immediate precursor of d-serine, a major agonist of the N-methyl-d-aspartate (NMDA) receptor. l-Serine is a pivotal amino acid since it serves as a precursor to a large number of essential metabolites besides d-serine. In all non-photosynthetic organisms, including mammals, a major source of l-serine is the phosphorylated pathway of l-serine biosynthesis.

The many faces of partial inhibition: Revealing imposters with graphical analysis.

Partial inhibition occurs when an enzyme-inhibitor complex is capable of producing a product, as opposed to complete inhibition where the enzyme-inhibitor complex is completely inactive and cannot produce a product. While not frequently encountered, partial inhibition is also not a rare phenomenon and can potentially have significant repercussions later on in a research project. It is therefore important to recognize partial inhibition when it occurs.

Elucidation of a Self-Sustaining Cycle in Escherichia coli l-Serine Biosynthesis That Results in the Conservation of the Coenzyme, NAD.

The equilibrium of the reaction catalyzed by d-3-phosphoglycerate dehydrogenase (PGDH), the first enzyme in the l-serine biosynthetic pathway, is far in the direction away from serine synthesis. As such, the enzyme is usually assayed in this direction. To easily assay it in the direction of l-serine synthesis, it can be coupled to the next enzyme in the pathway, phosphoserine aminotransferase (PSAT), with the activity monitored by the conversion of NAD to NADH by PGDH.

Regulatory Mechanism of Mycobacterium tuberculosis Phosphoserine Phosphatase SerB2.

Almost all organisms contain the same biosynthetic pathway for the synthesis of l-serine from the glycolytic intermediate, d-3-phosphoglycerate. However, regulation of this pathway varies from organism to organism. Many organisms control the activity of the first enzyme in the pathway, d-3-phosphoglycerate dehydrogenase (PGDH), by feedback inhibition through the interaction of l-serine with the ACT domains within the enzyme.

Modification of Cysteine.

This unit describes a number of methods for modifying cysteine residues of proteins and peptides. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N-ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N-(iodoethyl)-trifluoroacetamide are also presented.

Mutagenic and chemical analyses provide new insight into enzyme activation and mechanism of the type 2 iron-sulfur l-serine dehydratase from Legionella pneumophila.

The crystal structure of the Type 2 l-serine dehydratase from Legionella pneumophila (lpLSD), revealed a "tail-in-mouth" configuration where the C-terminal residue acts as an intrinsic competitive inhibitor. This pre-catalytic structure undergoes an activation step prior to catalytic turnover. Mutagenic analysis of residues at or near the active site cleft is consistent with stabilization of substrate binding by many of the same residues that interact with the C-terminal cysteine and highlight the critical role of certain tail residues in activity.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Unique Conformational and Chemical Transformations Occurring upon [4Fe-4S] Cluster Binding in the Type 2 L-Serine Dehydratase from Legionella pneumophila.

The type 2 L-serine dehydratase from Legionella pneumophila (lpLSD) contains a [4Fe-4S](2+) cluster that acts as a Lewis acid to extract the hydroxyl group of L-serine during the dehydration reaction. Surprisingly, the crystal structure shows that all four of the iron atoms in the cluster are coordinated with protein cysteinyl residues and that the cluster is buried and not exposed to solvent. If the crystal structure of lpLSD accurately reflects the structure in solution, then substantial rearrangement at the active site is necessary for the substrate to enter.

Comparison of Type 1 D-3-phosphoglycerate dehydrogenases reveals unique regulation in pathogenic Mycobacteria.

D-3-phosphoglycerate dehydrogenases (PGDH) from all organisms catalyze the conversion of D-3-phosphoglycerate to phosphohydroxypyruvate as the first step in the biosynthesis of l-serine. This investigation compares the properties of Type 1 PGDHs from seven different species and demonstrates that conserved residues in the ACT and ASB domains of some allow l-serine to act as a feedback inhibitor at low micromolar concentrations. In addition, the serine sensitivity is dependent on the presence of phosphate ions.

Structure of L-serine dehydratase from Legionella pneumophila: novel use of the C-terminal cysteine as an intrinsic competitive inhibitor.

Here we report the first complete structure of a bacterial Fe-S l-serine dehydratase determined to 2.25 Å resolution. The structure is of the type 2 l-serine dehydratase from Legionella pneumophila that consists of a single polypeptide chain containing a catalytic α domain and a β domain that is structurally homologous to the "allosteric substrate binding" or ASB domain of d-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis.

Regulation of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase by phosphate-modulated quaternary structure dynamics and a potential role for polyphosphate in enzyme regulation.

D-3-phosphoglycerate dehydrogenase (PGDH) catalyzes the first reaction in the "phosphorylated" pathway of l-serine biosynthesis. In Mycobacterium tuberculosis, it is a type 1 enzyme (mtPGDH) in that it contains both an ACT domain and an ASB domain in addition to a catalytic domain. The published crystal structures (Protein Data Bank entries 1YGY and 3DC2) show a tartrate molecule interacting with cationic residues at the ASB-ACT domain interfaces and a serine molecule bound at the ACT domain interface.

Identification and characterization of two new types of bacterial L-serine dehydratases and assessment of the function of the ACT domain.

Two new types of bacterial Fe-S L-serine dehydratases have been identified. These join two previously recognized enzyme types, for a total of four, that are distinguished on the basis of domain arrangement and amino acid sequence. A Type 3 enzyme from Amphibacillus xylanus (axLSD) and a Type 4 enzyme from Heliscomenobacter hydrossis (hhLSD) were cloned, expressed, purified, and characterized.

Kinetic evidence of a noncatalytic substrate binding site that regulates activity in Legionella pneumophila L-serine dehydratase.

The L-serine dehydratase from Legionella pneumophila (lpLSD) has recently been shown to contain a domain (β domain) that has a high degree of structural homology with the ASB domain of d-3-phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis. Furthermore, this domain has been shown by sequence homology to be present in all bacterial L-serine dehydratases that utilize an Fe-S catalytic center. In PGDH, L-serine binds to the ACT domain to inhibit catalytic activity.

Allosteric activation and contrasting properties of L-serine dehydratase types 1 and 2.

Bacterial L-serine dehydratases differ from mammalian L- and D-serine dehydratases and bacterial D-serine dehydratases by the presence of an iron-sulfur center rather than a pyridoxyl phosphate prosthetic group. They exist in two forms, types 1 and 2, distinguished by their sequence and oligomeric configuration. Both types contain an ASB domain, and the type 1 enzymes also contain an ACT domain in a tandem arrangement with the ASB domain like that in type 1 D-3-phosphoglycerate dehydrogenases (PGDHs).

Contrasting catalytic and allosteric mechanisms for phosphoglycerate dehydrogenases.

D-3-Phosphoglycerate dehydrogenases (PGDH) exist with at least three different structural motifs and the enzymes from different species display distinctly different mechanisms. In many species, particularly bacteria, the catalytic activity is regulated allosterically through binding of l-serine to a distinct structural domain, termed the ACT domain. Some species, such as Mycobacterium tuberculosis, contain an additional domain, called the "allosteric substrate binding" or ASB domain, that functions as a co-domain in the regulation of catalytic activity.

Kinetic, mutagenic, and structural homology analysis of L-serine dehydratase from Legionella pneumophila.

A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity.

Transient kinetic analysis of L-serine interaction with Escherichia coli D-3-phosphoglycerate dehydrogenase containing amino acid mutations in the hinge regions.

In Escherichia colid-3-phosphoglycerate dehydrogenase, the amino acid sequences G294-G295 and G336-G337 are found between structural domains and appear to function as hinge regions. Mutagenesis studies of these sequences showed that bulky side chains had significant effects on the kinetic properties of the enzyme. Placement of a tryptophanyl residue near the serine binding site (W139F/E360W) allows serine binding to be monitored by fluorescence quenching analysis.

Transient kinetic analysis of the interaction of L-serine with Escherichia coli D-3-phosphoglycerate dehydrogenase reveals the mechanism of V-type regulation and the order of effector binding.

Pre-steady state stopped-flow analysis of Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) reveals that the physiological inhibitor, l-serine, exerts its effect on at least two steps in the kinetic mechanism, but to very different degrees. First, there is a small but significant effect on the dissociation constant of NADH, the first substrate to bind in the ordered mechanism. The effect of serine is mainly on the binding off rate, increasing the K(d) to 5 and 23 muM from 0.

Role of the anion-binding site in catalysis and regulation of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase.

D-3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis displays substantial substrate inhibition in the direction of NADH oxidation by its physiological substrate, hydroxypyruvic acid phosphate (HPAP). Previous investigations showed that plots of substrate concentration versus activity derived from steady state assays could be fit with the equation for complete uncompetitive inhibition and that the mechanism may be allosteric. This investigation uses a simulation of transient kinetic data to demonstrate that the mechanism is consistent with the interaction of substrate at a second site called the anion-binding site.

A stopped flow transient kinetic analysis of substrate binding and catalysis in Escherichia coli D-3-phosphoglycerate dehydrogenase.

Pre-steady state, stopped flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior.

Structural analysis of substrate and effector binding in Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase.

The crystal structure of Mycobacterium tuberculosis d-3-phosphoglycerate dehydrogenase has been solved with bound effector, l-serine, and substrate, hydroxypyruvic acid phosphate, at resolutions of 2.7 and 2.4 A, respectively.

Synthetic peptides for production of antibodies that recognize intact proteins.

Antibodies that recognize intact proteins can be produced through the use of synthetic peptides based on short stretches of the protein sequence, without first having to isolate the protein. The steps to produce an effective antibody include: (1) designing the peptide sequence based on the sequence of the protein; (2) synthesizing the peptide; (3) preparing the immunogen either by coupling the synthetic peptide to a carrier protein or through the use of a multiple antigenic peptide (MAP); (4) immunizing the host animal; (5) assaying antibody titer in the host animal's serum; and (6) obtaining the antiserum and/or isolating the antibody. This unit covers steps 1 and 3.

Synthetic peptides for production of antibodies that recognize intact proteins.

Antibodies that recognize intact proteins can be produced through the use of synthetic peptides based on short stretches of the protein sequence, without first having to isolate the protein. The steps to produce an effective antibody include: (1) designing the peptide sequence based on the sequence of the protein; (2) synthesizing the peptide; (3) preparing the immunogen either by coupling the synthetic peptide to a carrier protein or through the use of a multiple antigenic peptide (MAP); (4) immunizing the host animal; (5) assaying antibody titer in the host animal's serum; and (6) obtaining the antiserum and/or isolating the antibody. This unit covers steps 1 and 3.

Synthetic peptides for production of antibodies that recognize intact proteins.

Antibodies that recognize intact proteins can be produced through the use of synthetic peptides based on short stretches of the protein sequence, without first having to isolate the protein. The procedure for selecting the antigenic sequence is relatively straightforward. This unit identifies ways to determine the best sequence and to chemically couple the synthetic peptide to a carrier protein to boost the immune response.