Characterization ofMycobacterium tuberculosisdiaminopimelic acid epimerase: paired cysteine residues are crucial for racemization

Abstract

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.

RppH-dependent pyrophosphohydrolysis of mRNAs is regulated by direct interaction with DapF in Escherichia coli

“…To study whether RppH activation by DapF is dependent on DapF enzymatic activity, the two catalytic cysteine residues of DapF, C73 and C217, were substituted to alanine (DapF(C73&217A)). Although the mutant protein did not catalyze the conversion of L,L-DAP to meso -DAP (42,43), it formed a tight complex with RppH and stimulated RppH activity (Figure 4). Because purified DapF(C73&217A) lacks the Ap 5 A hydrolase activity (Figure 2), the apparent activation of RppH by DapF(C73&217A) is unlikely to be due to contamination with RppH during the preparation of this protein.…”

Elucidation of the d‐lysine biosynthetic pathway in the hyperthermophile Thermotoga maritima

“…The substrate specificity of TM1522 for ll ‐Dpm and meso ‐Dpm is quite stringent, as indicated by our kinetic analysis (Table ). The K m values of TM1522 for ll ‐ or meso ‐Dpm ( K m for ll ‐Dpm = 0.082 m m ; K m for meso ‐Dpm = 0.258 m m ) were comparable to those of E. coli Dpm epimerase for ll ‐Dpm (0.129 m m ) and Mycobacterium tuberculosis Dpm epimerase for meso ‐Dpm (0.166 m m ) . The k cat value of TM1522 for ll ‐Dpm (56.2 s −1 ) was ~ 2‐fold lower than that of E. coli Dpm epimerase (120 s −1 ) , and the k cat value for meso ‐Dpm (79.1 s −1 ) was much higher than that of M. tuberculosis Dpm epimerase (0.147 s −1 ) .…”

Racemisation in Chemistry and Biology

“…Racemase (epimerase) enzymes that do not require pyridoxal include the diaminopimelic acid epimerase found in M. tuberculosis and proline racemase . It was found (by mutation and chemical transformation) that there are two key cysteine residues responsible for the stereochemical scrambling in these enzymes (Scheme ).…”

“…Reaction direction DG°(computed) DG°(experiment) [117] Review Racemase (epimerase) enzymes that do not require pyridoxal include the diaminopimelic acid epimerase found in M. tuberculosis and proline racemase. [118] It was found (by mutation and chemical transformation)t hat there are two key cysteine residuesr esponsible for the stereochemical scrambling in these enzymes (Scheme22). A computational study investigated the mechanism of action of proline racemase as well as the process that operates in aqueous solution.…”