Cloning and expression of streptomycin inactivating enzymes APH(6)-Ia and APH(6)-Id.

Discovered in the 1940s by Selman Waksman, the aminoglycoside antibiotic streptomycin is clinically important in the treatment of tuberculosis worldwide. However, strains of Mycobacterium tuberculosis and other pathogenic bacteria have become resistant to streptomycin. One mechanism by which this can occur is through the action of phosphotransferases that attach a phosphate group to position 6 of the streptidine ring of streptomycin, thereby inactivating it. Two such phosphotransferases are APH(6)-Ia from producer strain Streptomyces griseus, and APH(6)-Id found in animal, plant and human pathogenic isolates. Here, we report the subcloning and expression in Escherichia coli of soluble recombinant APH(6)-Ia and Id enzymes. Sequencing of aph(6)-Ia revealed a one-nucleotide disagreement with the published sequence, such that the amino acid at position 262 is an alanine instead of a serine. The sequence of aph(6)-Id is identical to that of the gene found in transposon Tn5393 of plant pathogen Erwinia amylovora. The successful expression of soluble forms of these enzymes now paves the way for experiments to study their structure and function by using site-directed mutagenesis.

Thermodynamics of reactions catalyzed by anthranilate synthase.

Microcalorimetry and high performance liquid chromatography have been used to conduct a thermodynamic investigation of reactions catalyzed by anthranilate synthase, the enzyme located at the first step in the biosynthetic pathway leading from chorismate to tryptophan. One of the overall biochemical reactions catalyzed by anthranilate synthase is: chorismate(aq) + ammonia(aq) = anthranilate(aq) + pyruvate(aq) + H2O(l). This reaction can be divided into two partial reactions involving the intermediate 2-amino-4-deoxyisochorismate (ADIC): chorismate(aq) + ammonia(aq) = ADIC(aq) + H2O(l) and ADIC(aq) = anthranilate(aq) + pyruvate(aq). The native anthranilate synthase and a mutant form of it that is deficient in ADIC lyase activity but has ADIC synthase activity were used to study the overall ammonia-dependent reaction and the first of the above two partial reactions, respectively. Microcalorimetric measurements were performed on the overall reaction at a temperature of 298.15 K and pH 7.79. Equilibrium measurements were performed on the first partial (ADIC synthase) reaction at temperatures ranging from 288.15 to 302.65 K, and at pH values from 7.76 to 8.08. The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate2-(aq) + NH4+(aq) = anthranilate-(aq) + pyruvate-(aq) + H+(aq) + H2O(l), delta rHmo = -(116.3 +/- 5.4) kJ mol-1. For the reaction: chorismate2-(aq) + NH4+(aq) = ADIC-(aq) + H2O(l), K = (20.3 +/- 4.5) and delta rHmo = (7.5 +/- 0.6) kJ mol-1. Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that are pertinent to this branch point of the chorismate pathway. The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes delta rG'mo under approximately physiological conditions are given.

Role of residue 161 in the allosteric transitions of two bacterial phosphofructokinases.

The loop between alpha-helix 6 and beta-strand F of the phosphofructokinase (PFK) from Bacillus stearothermophilus is proposed to be important in the allosteric transition of the enzyme [Schirmer, T., & Evans, P.R. (1990) Nature 343, 140-145]. Except for residue 161, the amino acids within the loop are similar between B. stearothermophilus PFK (BsPFK) and the PFK from Escherichia coli (EcPFK). In the former enzyme, residue 161 is a glutamate, while in the latter it is a glutamine. We have used site-directed mutagenesis to investigate the importance of residue 161 for the allosteric regulation of the two enzymes by phosphoenolpyruvate (PEP), an inhibitor, and GDP, an activator. In BsPFK, glutamate 161 has been changed to a glutamine and an alanine, while in EcPFK, glutamine 161 has been changed to a glutamate, an arginine, and an alanine. The kinetic parameters of the mutant enzymes were similar to those of the respective wild types, indicating that residue 161 is not directly involved in substrate binding and catalysis. One of the EcPFK mutants, Q161A, though activated normally by GDP, was completely insensitive to PEP. This indicates that the hydrogen-bonding ability of residue 161 is critical for PEP inhibition of EcPFK and suggests that GDP activation and PEP inhibition follow different structural pathways in EcPFK. The BsPFK mutant enzymes were less sensitive to PEP inhibition and more sensitive to GDP activation, suggesting that inhibition and activation are opposed and follow a common structural pathway in agreement with a concerted allosteric mechanism.

A chimeric bacterial phosphofructokinase exhibits cooperativity in the absence of heterotropic regulation.

The phosphofructokinases (PFKs) from the bacteria Escherichia coli and Bacillus stearothermophilus differ markedly in their regulation by ATP. Whereas E. coli PFK (EcPFK) is profoundly inhibited by ATP, B. stearothermophilus PFK (BsPFK) is only slightly inhibited. The structural basis for this difference could be closure of the active site via a conformational transition that occurs in the ATP-binding domain of EcPFK, but is absent in BsPFK. To investigate the role of this transition in ATP inhibition of EcPFK, we have constructed a chimeric enzyme that contains the "rigid" ATP-binding domain of BsPFK grafted onto the remainder of the EcPFK subunit. The chimeric PFK has the following characteristics: (i) tetrameric structure and kinetic parameters similar to those of the native enzymes, (ii) insensitivity to regulation by the effector phosphoenolpyruvate despite its ability to bind to the enzyme, and (iii) a sigmoidal (nH around 2) fructose 6-phosphate saturation curve. From the results, it is concluded that the active site regions of the two native enzymes are remarkably similar, but their effector sites and their mechanisms of heterotropic regulation are different. The chimeric subunit is locked in a structure resembling that of activated E. coli PFK, yet the enzyme can exist in two different conformational states. Mechanisms for its sigmoidal kinetics are discussed.