Lambda lysozyme, overexpression, ligand studies, and incorporation of trifluoromethionine

Abstract

Lysozymes are widely distributed in nature and function to cleave the glycan chains of the bacterial peptidoglycan. The lysozyme from bacteriophage lambda (lambda lysozyme; LaL) is the late gene product of the lambda R gene. the lambda R gene has been incorporated into several plasmids for the intended high level expression of LaL in Eschericia coli. An efficient procedure permitting purification of LaL from three of the four expression systems was established. The highest level of overproduction of LaL, producing 25-30 mg of purified LaL per litre of culture, was achieved by the expression of LaL from E. coliBL2 l(WE3) harboring plasmid pLR102.

A turbidimetric assay utilizing chloroform-sensitized E. coli substrate cells has been optimized to assess the bacteriolytic activity of LaL. Several preparations of the substrate cells have indicated a "batch to batch• variation with respect to their susceptibility to LaL. Chitooligosaccharides ((GlcNAc)o; oligomers of P-1,4-N-acet;yl-D glucosemine) were shown to inhibit the bacteriolytic activity of LaL but were not cleaved by the enzyme. Differential scanning calorimetry revealed that the temperature at which the thermal denaturation of LaL occurred increased in the presence of (GlcNAc)3 and (GlcNAc)s. Lambda lysozyme was also expressed in an E. coli strain auxotrophic for methionine, allowing for the incorporation of (methyl-13C)methionine in the enzyme. An alteration of the [1H-13C]HMQC NMR spectra of the labelled enzyme was observed in the presence of (GlcNAc)s. Chemical modification studies suggested the importance of acidic residue(s) for the bacteriolytic activity of LaL and chitooligosaccharides were shown to protect carboxyl group modification. The observed pattern of inhibition and carboxyl group protection by chitooligosaccbandes and the effect of cbitooligosaccberides on the intrinsic fluorescence of LaL suggested a possible difference in the binding modes for (GlcNAc)~ and for (GlcNAc)IQ:4 to LaL. The results indicate that LaL has specific interactions with oligosaccharides of N-acetylglucnsamine but is incapable of cleaving the glycosidic bonds of these saccharides.

Two small peptides (L-Ala-D-wo-Glu-L-Lys-(D-Ala)a, n• 1 or 2) mimicking the peptide portion of E. coli peptidoglycan were prepared and their interactions with LaL were evaluated. Affinity capillary electrophoresis suggested the possibility of a complex formation between peptide and LaL. Interestingly, the bacteriolytic activity of LaL was found to increase in the presence of the peptides. In addition, evidence is presented suggesting that only in the presence of exogenous peptide is LaL capable of cleaving a chitooligosaccbande substrate. The synthesis of p-nitrophenyl-P-GlcNAc (P-PNP-GlcNAc) and some substituted derivatives of P-PNP-N-acefylmuramic acid is described. The muramic acid analogues did not serve as substrates for LaL or hen's egg white lysozyme.

Much interest is currently focused on understanding the detailed contribution that particular amino acid residues make in protein structure and function. Fluorinated amino acids have been utilized successfully to probe protein structure and dynamics and have pointed to the importance of specific residues to biological function. The importance of the amino acid methionine in proteins has been examined by the successful incorporation of LS- trifluoromethylhomocysteine (L-tmluorometbionine; L TFM) into lambda lysozyme, an enzyme containing three methionine residues. The L isomer of TFM was synthesized in an overall yield of 33% from N-acefyl-D, L-homocysteine tbiolactone and trifluoromethyl iodide. The expression plasmid pLR102 was transformed into an E. coli strain auxotrophic for methionine permitting the expression of LaL in the presence of L-TFM. The analogue would not support growth of the auxotroph and was found to be inlul>itory to cell growth. However, cells that were initially grown in a methionine rich media followed by protein induction under careful control of the respective concentrations of L metbionine and LTFM in the media, were able to overexpress TFM-labelled LaL (TFM-LaL) at both high (70 and 74%) and low (31 %) levels ofTFM incorporation. TFM-LaL at both levels of incorporation extu.bited activity analogous with that of the wild type enzyme and were inhibited by cbitooligosaccharides indicating that replacement of the methionine residues by TFM did not hinder enzyme function. Interestingly, the 19F solution NMR spectra of the TFM labelled enzymes consisted of four sharp resonances spanning a chemical shift range of 0.9 ppm, with three of the resonances showing very modest shielding changes on binding of (GlcNAc)s. The 19F NMR analysis of TFM-LaL at both mgh. and low levels of incorporation suggested that one of the methionine positions gives rise to two separate resonances. The intensities of these two resonances were influenced by the extent of incorporation which was interpreted as an indication that subtle conformational changes in protein structure accompany the incorporation of TFM. The similarities and differences between Met and TFM were analyzed using semi-empirical and ab initio molecular orbital calculations. The TFM-labelled enzymes were found to be unreactive towards chemical cleavage with cyanogen bromide. Mass spectral analysis has strongly suggested the specific formylation of the hydroxyl groups of serine, threonine and C-tenninal homoserine residues during the course of the cyanogen bromide reaction. The methodology presented offers promise as a new approach to the study of protein-ligand interactions as well as for future investigations into the functional importance of methionine in proteins.