A critical examination of Escherichia coli esterase activity

Abstract

The ability of Escherichia coli to grow on a series of acetylated and glycosylated compounds has been investigated. It is surmised that E. coli maintains low levels of nonspecific esterase activity. This observation may have ramifications for previous reports that relied on nonspecific esterases from E. coli to genetically encode nonnatural amino acids. It had been reported that nonspecific esterases from E. coli deacetylate tri-acetyl O-linked glycosylated serine and threonine in vivo. The glycosylated amino acids were reported to have been genetically encoded into proteins in response to the amber stop codon. However, it is our contention that such amino acids are not utilized in this manner within E. coli. The current results report in vitro analysis of the original enzyme and an in vivo analysis of a glycosylated amino acid. It is concluded that the amber suppression method with nonnatural amino acids may require a caveat for use in certain instances.

FIGURE 1.

Proposed product of an esterase with GlcNAc-Ser and other esterase substrates discussed in this study.

FIGURE 2.

Growth curves of E. coli on various carbon sources (1% v/v or w/v) in the presence and absence of porcine liver esterase (1760 units/liter). Abs, absorbance.

FIGURE 3.

Growth of E. coli on tri-acetyl-β-GlcNAc-serine, glycerol, and triacetin as the sole carbon sources in minimal media in the presence and absence of porcine liver esterase (4000 units/liter). Cultures were measured at 15 h, and all carbon sources provided as 0.5% v/v or w/v.

FIGURE 4.

Examination of E. coli esterase activity. The experiment requires hydrolysis of a methyl ester of p-iodophenylalanine to complement the phenotype of an amber mutant of GFP. Identical cultures were supplemented with 1 mm free and protected amino acid as indicated and incubated 24 h.

FIGURE 5.

Isothermal calorimetry measurements. A, results for p-acetylphenylalanine with a mutant aminoacyl tRNA synthetase (aaRS^keto) evolved to be specific for the amino acid (36). B, results for tyrosine with aminoacyl tRNA synthetase (aaRS^keto) evolved for p-acetylphenylalanine. C, results for tyrosine with the (S)1–90 aaRS reported by Zhang et al. (1). D, results for GlcNAc-Ser with the (S)1–90 aaRS reported by Zhang et al. (1).