Role of paired basic residues in the expression of active recombinant galactosyltransferases from the bacterial pathogen Neisseria meningitidis

Abstract

The lgtB gene encoding a beta-1,4-galactosyltransferase gene and the lgtC gene encoding an alpha-1,4-galactosyltransferase from the bacterial pathogen Neisseria meningitidis were cloned into an expression vector and overexpressed in Escherichia coli. Both genes expressed very well, but problems with C-terminal proteolysis were encountered with both proteins. The lgtC protein was initially isolated from extracts of recombinant E.coli as a truncated species that retained enzymatic activity, and was subsequently shown by mass spectrometry to be 19 residues shorter than the expected protein. A specific set of engineered C-terminal deletions was constructed to investigate their effect on the expression of lgtC. As many as 28 residues could be deleted with little effect on activity, and with the concomitant improvement of the overall expression up to fivefold over the full length protein. The lgtB protein was also proteolysed in extracts of normal E.coli strains into enzymatically inactive fragments lacking 28 or 41 C-terminal residues. This degradation could be prevented by expression in an ompT protease deficient strain of E.coli. The full length lgtB protein was not stable in soluble protein extracts from all recombinant strains, however a stable enzyme preparation could be achieved with the membrane fraction from cells of the ompT deficient strain expressing lgtB. Specific deletions of lgtB were also constructed, and 15 residues could be removed without loss of enzyme activity and also with the concomitant improvement of the overall expression up to twofold over the full length protein. Longer deletions produced protein but activity could not be detected in these recombinant strains. Examination of the glycosyltransferase sequences from a wide range of bacteria showed their C-terminal segments of approximately 50 amino acids frequently contained paired basic residues. Engineering of these segments may therefore be required as a general practice to produce these enzymes for use in the large scale chemi-enzymatic synthesis of carbohydrate-based therapeutics.

Glycosyl Fluorides Can Function as Substrates for Nucleotide Phosphosugar-dependent Glycosyltransferases

“…␣-Galactosyl fluoride was first tested as a donor substrate for the recombinant lipopolysaccharide galactosyltransferase C (lgtCϪ19) from N. meningitidis (36) in the presence of several readily-detected fluorescent galactosides as acceptors. When reaction mixtures containing ␣-galactosyl fluoride plus, individually, FITC lac, FCHASE gal, and FCHASE lac were incubated with lgtCϪ19 in buffer containing Mn 2ϩ and DTT, no reaction product could be detected after 1 h by thin-layer chromatography or HPLC analysis, or by use of a fluoride electrode.…”

“…General Procedures-Recombinant ␣-galactosyltransferase from Neisseria meningitidis lacking the 19 C-terminal amino acid residues was expressed in, and purified from, Escherichia coli as described previously (36). Concentrations of lgtCϪ19 solutions were determined based on ⑀ 280 ϭ 1.74 ml mg Ϫ1 cm Ϫ1 (36).…”

“…Concentrations of lgtCϪ19 solutions were determined based on ⑀ 280 ϭ 1.74 ml mg Ϫ1 cm Ϫ1 (36). Type II rabbit muscle pyruvate kinase and lactate dehydrogenase were obtained from Sigma as suspensions in 3.2 M ammonium sulfate, pH 6.0.…”

Intermediate Trapping on a Mutant Retaining α-Galactosyltransferase Identifies an Unexpected Aspartate Residue

“…UDP-galactose was a generous gift from Neose Inc. Recombinant LgtC was overexpressed and purified from Escherichia coli (BL21) as described previously (26).…”

Fungal trehalose phosphorylase: kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune

“…Water molecules complete the co-ordination sphere of the metal ion. In the case of galactosyltransferase [27], Mn# + has been shown to be required for the stability of the enzyme fold [39]. Interestingly, a very acidic Asp-Asp-Ile(Leu)-Asp(Glu)-Glu motif is found in all three known trehalose phosphorylases (Table 4 ; positions 240-251 in the G. frondosa enzyme).…”