Inorganic polyphosphate (poly P) is a chain of tens or many hundreds of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. Despite inorganic polyphosphate's ubiquity--found in every cell in nature and likely conserved from prebiotic times--this polymer has been given scant attention. Among the reasons for this neglect of poly P have been the lack of sensitive, definitive, and facile analytical methods to assess its concentration in biological sources and the consequent lack of demonstrably important physiological functions. This review focuses on recent advances made possible by the introduction of novel, enzymatically based assays. The isolation and ready availability of Escherichia coli polyphosphate kinase (PPK) that can convert poly P and ADP to ATP and of a yeast exopolyphosphatase that can hydrolyze poly P to Pi, provide highly specific, sensitive, and facile assays adaptable to a high-throughput format. Beyond the reagents afforded by the use of these enzymes, their genes, when identified, mutated, and overexpressed, have offered insights into the physiological functions of poly P. Most notably, studies in E. coli reveal large accumulations of poly P in cellular responses to deficiencies in an amino acid, Pi, or nitrogen or to the stresses of a nutrient downshift or high salt. The ppk mutant, lacking PPK and thus severely deficient in poly P, also fails to express RpoS (a sigma factor for RNA polymerase), the regulatory protein that governs > or = 50 genes responsible for stationary-phase adaptations to resist starvation, heat and oxidant stresses, UV irradiation, etc. Most dramatically, ppk mutants die after only a few days in stationary phase. The high degree of homology of the PPK sequence in many bacteria, including some of the major pathogenic species (e.g. Mycobacterium tuberculosis, Neisseria meningitidis, Helicobacter pylori, Vibrio cholerae, Salmonella typhimurium, Shigella flexneri, Pseudomonas aeruginosa, Bordetella pertussis, and Yersinia pestis), has prompted the knockout of their ppk gene to determine the dependence of virulence on poly P and the potential of PPK as a target for antimicrobial drugs. In yeast and mammalian cells, exo- and endopolyphosphatases have been identified and isolated, but little is known about the synthesis of poly P or its physiologic functions. Whether microbe or human, all species depend on adaptations in the stationary phase, which is truly a dynamic phase of life. Most research is focused on the early and reproductive phases of organisms, which are rather brief intervals of rapid growth. More attention needs to be given to the extensive period of maturity. Survival of microbial species depends on being able to manage in the stationary phase. In view of the universality and complexity of basic biochemical mechanisms, it would be surprising if some of the variety of poly P functions observed in microorganisms did not apply to aspects of human growth and development, to aging, and to the aberrations of disease. Of theoretical interest regarding po...

A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP. We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light. This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein. A simplified procedure for preparing polyP synthesized by polyP kinase is also described. Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP. By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels. Analysis of Escherichia coli mutants revealed that polyP accumulation depends on several regulatory genes, glnD (NtrC), rpoS,relA, and phoB.

Total chemical synthesis was used to prepare the mirror image ( D -protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D -VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D - and L - forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L -protein ligand bound only to D -VEGF-A, whereas the D -protein ligand bound only to L -VEGF-A. The D -protein ligand, but not the L -protein ligand, inhibited the binding of natural VEGF 165 to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of { D -protein antagonist +  L -protein form ofVEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D -protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800  Å 2 in accord with our design objectives, and that the D -protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

Polypeptides composed entirely of d-amino acids and the achiral amino acid glycine (d-proteins) inherently have in vivo properties that are proposed to be near-optimal for a large molecule therapeutic agent. Specifically, d-proteins are resistant to degradation by proteases and are anticipated to be nonimmunogenic. Furthermore, d-proteins are manufactured chemically and can be engineered to have other desirable properties, such as improved stability, affinity, and pharmacokinetics. Thus, a well-designed d-protein therapeutic would likely have significant advantages over l-protein drugs. Toward the goal of developing d-protein therapeutics, we previously generated RFX001.D, a d-protein antagonist of natural vascular endothelial growth factor A (VEGF-A) that inhibited binding to its receptor. However, RFX001.D is unstable at physiological temperatures (Tm = 33 °C). Here, we describe RFX037.D, a variant of RFX001.D with extreme thermal stability (Tm > 95 °C), high affinity for VEGF-A (Kd = 6 nM), and improved receptor blocking. Comparison of the two enantiomeric forms of RFX037 revealed that the d-protein is more stable in mouse, monkey, and human plasma and has a longer half-life in vivo in mice. Significantly, RFX037.D was nonimmunogenic in mice, whereas the l-enantiomer generated a strong immune response. These results confirm the potential utility of synthetic d-proteins as alternatives to therapeutic antibodies.

Human placental S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) was inactivated by 5,5-dithiobis(2-nitrobenzoic acid) following pseudo-first-order kinetics. Modification of three of the 10 cysteine residues per enzyme subunit resulted in complete inactivation of the enzyme.