Identification and functional analysis of Escherichia coli cysteine desulfhydrases

Abstract

In Escherichia coli, three additional proteins having L-cysteine desulfhydrase activity were identified as O-acetylserine sulfhydrylase-A, O-acetylserine sulfhydrylase-B, and MalY protein, in addition to tryptophanase and cystathionine beta-lyase, which have been reported previously. The gene disruption for each protein was significantly effective for overproduction of L-cysteine and L-cystine. Growth phenotype and transcriptional analyses suggest that tryptophanase contributes primarily to L-cysteine degradation.

FIG. 1.

Detection of OASS-A, MalY, and OASS-B by CD activity staining. Preparation of native PAGE gel, cell extracts, and CD activity staining were carried out according to the method described previously (2). Lane 1, wild-type JM39 harboring pBluescript II SK+ (vector control; Toyobo Biochemicals, Osaka, Japan); lane 2, JM39 harboring pcysK (cysK plasmid; OASS-A is overexpressed); lane 3, JM39 harboring pcysM (cysM plasmid; OASS-B is overexpressed); lane 4, JM39 harboring pmalY (malY plasmid; MalY is overexpressed).

FIG. 2.

TNase induction by the addition of l-cysteine. Total RNA of JM39 was prepared from cells cultivated in LB medium (lane 1) and LB plus 10 mM l-cysteine (lane 2). Each lane was loaded with 20 μg of total RNA. The arrow indicates a tnaC-tnaA transcript of ca. 1.7 kb. The 23S and 16S rRNAs are total RNA-loading controls detected by UV spectrometer.