Leisha S. Mullins

D-Ala-D-X ligases: evaluation of D-alanyl phosphate intermediate by MIX, PIX and rapid quench studies

BACKGROUND The D-alanyl-D-lactate (D-Ala-D-Lac) ligase is required for synthesis of altered peptidoglycan (PG) termini in the VanA phenotype of vancomycin-resistant enterococci (VRE), and the D-alanyl-D-serine (D-Ala-D-Ser) ligase is required for the VanC phenotype of VRE. Here we have compared these with the Escherichia coli D-Ala-D-Ala ligase DdlB for formation of the enzyme-bound D-alanyl phosphate, D-Ala(1)-PO(3)(2-) (D-Ala(1)-P), intermediate. RESULTS The VanC2 ligase catalyzes a molecular isotope exchange (MIX) partial reaction, incorporating radioactivity from (14)C-D-Ser into D-Ala-(14)C-D-Ser at a rate of 0.7 min(-1), which approaches kinetic competence for the reversible D-Ala(1)-P formation from the back direction. A positional isotope exchange (PIX) study with the VanC2 and VanA ligases displayed a D-Ala(1)-dependent bridge to nonbridge exchange of the oxygen-18 label of [gamma-(18)O(4)]-ATP at rates of up to 0.6 min(-1); this exchange was completely suppressed by the addition of the second substrate D-Ser or D-Lac, respectively, as the D-Ala(1)-P intermediate was swept in the forward direction. As a third criterion for formation of bound D-Ala(1)-P, we conducted rapid quench studies to detect bursts of ADP formation in the first turnover of DdlB and VanA. With E. coli DdlB, there was a burst amplitude of ADP corresponding to 26-30% of the DdlB active sites, followed by the expected steady-state rate of 620-650 min(-1). For D-Ala-D-Lac and D-Ala-D-Ala synthesis by VanA, we measured a burst of 25-30% or 51% of active enzyme, respectively. CONCLUSIONS These three approaches support the rapid (more than 1000 min(-1)), reversible formation of the enzyme intermediate D-Ala(1)-P by members of the D-Ala-D-X (where X is Ala, Ser or Lac) ligase superfamily.

Effect of linker sequence on the stability of circularly permuted variants of ribonuclease T1

Circularly permuted variants of ribonuclease T1 were constructed with a library of residues covalently linking the original amino and carboxyl terminal ends of the wild-type protein. The library of linking peptides consisted of three amino acids containing any combination of proline, aspartate, asparagine, serine, threonine, tyrosine, alanine, and histidine. Forty two unique linker sequences were isolated and 10 of these mutants were further characterized with regard to catalytic activity and overall thermodynamic stability. The 10 mutants with the different linking sequences (HPD, TPH, DTD, TPD, PYH, PAT, PHP, DSS, SPP, and TPS), in addition to GGG and GPG, were 4.0-6.2 kcal/mol less stable than the wild-type ribonuclease T1. However, these circular permuted variants were only 0.4-2.6 kcal/mol less stable than the direct parent protein that is missing the disulfide bond connecting residues 2 and 10. The most stable linking peptide was HPD.

Circular permutation of RNase T1 through PCR based site-directed mutagenesis

Publisher Summary Current methods for creating circularly permuted variants through alterations in the genetic sequence of a protein are laborious and involve a large number of recombinant DNA manipulations. This chapter discusses the utility of a general PCR approach for the creation of circularly permuted proteins through the initial characterization of cp35SL, and discusses the construction of a circularly permuted variant of ribonuclease Tl (RNase Tl). The native RNase Tl, (C2A, ClOA), and cp35Sl proteins are characterized by specific activity, polyacrylamide electrophoresis (SDS and native), thermodynamic stability, matrix-assisted laser desorption ionization (MALDI) mass spectrometry, and amino-terminal sequencing. The specific activity and thermodynamic stability of the (C2A, ClOA) mutant confirm that the Cys-2 to Cys-10 disulfide bond imparts thermodynamic stability but has little effect on catalytic activity. Hence this mutant was selected as the starting point for constructing a circularly permuted form of RNase-Tl, such that as short a linker as possible could be used to bridge the original N- and C-termini. The activity and stability of the circularly permuted variant indicate that, it adopts an overall tertiary fold very similar to that of the native protein. However, the real effect, however, may be more evident in the kinetics of the specific folding pathway.

Phosphorus-31 NMR relaxation studies of diethyl P-methoxyphenyl phosphate bound to phosphotriesterase

Abstract The effect of Mn 2+ Mn 2+ , Mn 2+ Zn 2+ and Mn 2+ Cd 2+ reconstituted phosphotriesterase on the 31P spin lattice ( 1 T 1 ) relaxation rate of diethyl p-methoxyphenyl phosphate has been investigated. In the presence of Mn 2+ Mn 2+ phosphotriesterase, the spin lattice relaxation rate of the phosphorus atom is enhanced giving an upper limit for the phosphorus-metal root mean-sixth average distance of 4.2 A. These results demonstrate for the first time that substrates for phosphotriesterase bind in close proximity to the binuclear metal center.