B. M. Wöhrl

Kinetic Analysis of Four HIV-1 Reverse Transcriptase Enzymes Mutated in the Primer Grip Region of p66 IMPLICATIONS FOR DNA SYNTHESIS AND DIMERIZATION

The highly conserved primer grip region in the p66 subunit of HIV-1 reverse transcriptase (RT) is formed by the β12-β13 hairpin (residues 227–235). It has been proposed to play a role in aligning the 3′-OH end of the primer in a position for nucleophilic attack on an incoming dNTP. To analyze the importance of the primer grip for RT function, mutant RTs were used that contain single alanine substitutions of residues Trp229, Met230, Gly231, and Tyr232 in the p66 subunit of the heterodimeric p66/51 enzyme. Steady-state and pre-steady-state kinetic analyses of the enzymes were performed. All mutant enzymes revealed reduced polymerase activity. Mutation of Y232A showed the smallest effect on polymerase function. Equilibrium fluorescence titrations demonstrated that the affinity of the mutants for tRNA was only slightly affected. However, the affinity for primer-template DNA was reduced 27-fold for mutant p66W229A/51 and 23-fold for mutant p66G231A/51, and the maximal pre-steady-state rate of nucleotide incorporation,k pol, was reduced 27-fold for p66W229A/51 and 70-fold for p66G231A/51, respectively. Mutant p66M230A/51 revealed no reduced affinity for primer-template but showed a 71-fold reduced affinity for dTTP. Additionally, the mutations Trp229 and Gly231 affected the stability of the RT heterodimer.

Molecular analysis of the phosphoenolpyruvate-dependent L-sorbose: phosphotransferase system from Klebsiella pneumoniae and of its multidomain structure.

We have cloned a 3.4 kb DNA fragment from the chromosome of Klebsiella pneumoniae that codes for a phosphoenolpyruvate-dependent l-sorbose: phosphotransferase system (PTS). The cloned fragment was sequenced and four open reading frames coding for 135 (sorF), 164 (sorB), 266 (sorA) and 274 (sorM) amino acids, respectively, were found. The corresponding proteins could be detected in a T7 overexpression system, which yielded molecular masses of about 14000 for SorF, 19000 for SorB, 25000 for SorA and 27000 for SorM. SorF and SorB have all the characteristics of soluble and intracellular proteins in accordance with their functions as EIIASor and EIIBSor domains of the l-sorbose PTS. SorA and SorM, by contrast, are strongly hydrophobic, membrane-bound proteins with two to five putative transmembrane helices that alternate with a series of hydrophilic loops. They correspond to domains EIICSor and EIIDSor. The four proteins of the l-sorbose PTS resemble closely (27%–60%) the four subunits of a d-fructose PTS (EIIALev, EIIBLev, EIICLev, and EIIDLev) from Bacillus subtilis and the three subunits of the d-mannose PTS (EIIA,BMan, EIICMan, and EIIDMan) from Escherichia coli K-12. The three systems constitute a new PTS family, and sequence comparisons revealed highly conserved structures for the membranebound proteins. A consensus sequence for the membrane proteins was used to postulate a model for their integration into the membrane.