Characterization of the secreted chorismate mutase from the pathogen Mycobacterium tuberculosis

Abstract

The gene encompassing ORF Rv1885c with weak sequence similarity to AroQ chorismate mutases (CMs) was cloned from the genome of Mycobacterium tuberculosis and expressed in Escherichia coli. The gene product (*MtCM) complements a CM‐deficient E. coli strain, but only if produced without the predicted N‐terminal signal sequence typical of M. tuberculosis. The mature *MtCM, which was purified by exploiting its resistance to irreversible thermal denaturation, possesses high CM activity in vitro. The enzyme follows simple Michaelis–Menten kinetics, having a kcat of 50 s−1 and a Km of 180 µm (at 30 °C and pH 7.5). *MtCM was shown to be a dimer by analytical ultracentrifugation and size‐exclusion chromatography. Secondary‐structure prediction and CD spectroscopy confirmed that *MtCM is a member of the all‐α‐helical AroQ class of CMs, but it seems to have a topologically rearranged AroQ fold. Because CMs are normally intracellular metabolic enzymes required for the biosynthesis of phenylalanine and tyrosine, the existence of an exported CM in Gram‐positive M. tuberculosis is puzzling. The observation that homologs of *MtCM with a predicted export sequence are generally only present in parasitic or pathogenic organisms suggests that secreted CMs may have evolved to participate in some aspect of parasitism or pathogenesis yet to be unraveled.

Mechanistic Insights into the Isochorismate Pyruvate Lyase Activity of the Catalytically Promiscuous PchB from Combinatorial Mutagenesis and Selection

“…The positive control plasmid pKIL10A-W also encodes PchB H . Gene expression is under dual-promoter control (23,29), either from the salicylate-dependent promoter P sal (33) or from the P T7 promoter/lac operator sequence. pKIL10A-W was constructed by ligating the 4,124-bp NheI-SacII fragment of pKIL10A-0 and the 164-bp NheI/ SacII-digested PCR fragment generated on template pKSS-TB with primers 118-LWTS (5Ј-GTCAGAGCTAGCGAGGCaGCGATTCCGGCGCCaGAGCGGGTCGCaGCGATGCTCCCCGAGCG-3Ј) and 119-LWTN (5Ј-GTCAAACCGCGGGTCTGaCGCCAGTACTTGATCTGCTCaGCGATGTACCAGTGGATGATCTGCGCGAACAG-TC-3Ј) (lowercase letters indicate silent mutations introduced to optimize codons for high gene expression in E. coli (34)).…”

Design, selection, and characterization of a split chorismate mutase

“…However, mixing the components at equimolar concentrations (0.1 lM) produced no significant CM activity in vitro (data not shown). Furthermore, when the genes for MjCM * 22-93 and a representative variant (Glu3Gly) of MjCM were subcloned into the selection plasmid pMG211 28 (yielding pMG211B_split, see Materials and Methods), coexpression in the CM-deficient E. coli selection strain KA12/pKIMP-UAUC 29 did not allow for growth on selective agar plates lacking phenylalanine and tyrosine (Supporting information Fig. 1).…”

“…The charged glutamate and lysine residues in Z-C3 were retained from the design of mMjCM. 18 The genes for both polypeptides were assembled by overlap extension PCR from a plasmid encoding N1-ZÁZ-C3 with randomized primers, and ligated into a derivative of pMG211 28 (Supporting information Fig. 3).…”

“…5). pMG211-mMjCM was prepared by ligating the 4561 bp NdeI-XhoI fragment of pMG211 28 and the 306 bp NdeI-XhoI fragment of pET-mMjCM 0 , which was constructed by inserting the sequence for the selected monomer into pET22b(þ) 18 (Andreas Kleeb, unpublished). The DNA fragment for the truncated MjCM 22-93 was amplified from pMG211-mMjCM using primers CM2293f and CM2293r and digested with NdeI and SpeI yielding a 225 bp fragment, which was ligated into the 4529 bp fragment from NdeI and SpeI-digested pMG211_mMjCM, yielding pMG211_mMjCM .…”

“…28 All measurements were performed in PBS at pH 6.5 and 25 C in the presence of 0.1 mg/mL BSA. For Michaelis-Menten kinetics at 0.1 lM enzyme concentration, hdCM was preequilibrated as a 10 lM stock solution in the presence of 1 mg/mL BSA in PBS, pH 6.5, in a 1.5 mL Eppendorf tube for several minutes.…”