Enzymatic Characterization of Dihydrolipoamide Dehydrogenase from Streptococcus pneumoniae Harboring Its Own Substrate

Abstract

This study describes the enzymatic characterization of dihydrolipoamide dehydrogenase (DLDH) from Streptococcus pneumoniae and is the first characterization of a DLDH that carries its own substrate (a lipoic acid covalently attached to a lipoyl protein domain) within its own sequence. Full-length recombinant DLDH (rDLDH) was expressed and compared with enzyme expressed in the absence of lipoic acid (rDLDH ؊LA ) or with enzyme lacking the first 112 amino acids constituting the lipoyl protein domain (rDLDH ؊LIPOYL ). All three proteins contained 1 mol of FAD/mol of protein, had a higher activity for the conversion of NAD ؉ to NADH than for the reaction in the reverse direction, and were unable to use NADP ؉ and NADPH as substrates. The enzymes had similar substrate specificities, with the K m for NAD ؉ being ϳ20 times higher than that for dihydrolipoamide. The kinetic pattern suggested a Ping Pong Bi Bi mechanism, which was verified by product inhibition studies. The protein expressed without lipoic acid was indistinguishable from the wild-type protein in all analyses. On the other hand, the protein without a lipoyl protein domain had a 2-3-fold higher turnover number, a lower K I for NADH, and a higher K I for lipoamide compared with the other two enzymes. The results suggest that the lipoyl protein domain (but not lipoic acid alone) plays a regulatory role in the enzymatic characteristics of pneumococcal DLDH.Dihydrolipoamide dehydrogenase (DLDH 2 ; EC 1.8.1.4) is a flavoenzyme that constitutes the E3 component or L protein of five characterized 2-oxoacid dehydrogenase complexes (1-4). DLDH is also anticipated to have other functions, as it is present in organisms that do not contain 2-oxoacid dehydrogenase complexes (5-7). In Escherichia coli, DLDH stimulates ATPbinding cassette transport of several carbohydrates (8, 9) and ubiquinone-mediated transport of amino acids (10); in fission yeast, it is involved in cell cycle progression (11); and in Neisseria meningitidis, DLDH constitutes an immunogenic surface antigen (12, 13). Whether enzymatic activity is required to perform these functions is not known.In a 2-oxoacid dehydrogenase complex, the DLDH enzyme catalyzes the terminal pyridine nucleotide-linked reoxidation of a protein-bound dihydrolipoic acid using NAD ϩ as an electron acceptor and FAD as a prosthetic group (4). Purified DLDH is capable of catalyzing the reversible NAD ϩ -dependent oxidation of free dihydrolipoamide and other reduced lipoic acid derivatives (14 -16). The kinetic mechanism of DLDH has been well characterized using enzyme preparations from mammalian cells, yeast, Escherichia coli, and mycobacteria (4,(17)(18)(19)(20)(21)(22)(23)(24). Catalysis by the DLDH enzyme occurs via a Ping Pong Bi Bi reaction mechanism, which has been verified by both substrate inhibition kinetics and isotope exchange between NADH and NAD ϩ (18,21,22). Furthermore, the enzyme shows a strong substrate inhibition by NADH, making it difficult to measure the initial velocity of the forward reaction.We recently ident...

Lipoic Acid Metabolism in Microbial Pathogens

“…This domain is instead found at the N terminus of the dihydrolipoamide dehydrogenase (DLDH) (210), where it appears to regulate the activity of this enzyme (75). DLDH enzymes with N-terminal lipoyl domains are also found in several other species (Tables 2 and 4), including Clostridium magnum (115), C. difficile, C. botulinum, S. pyogenes, N. gonorrhoeae, and N. meningitidis (210).…”

Ohr (Organic Hydroperoxide Resistance Protein) Possesses a Previously Undescribed Activity, Lipoyl-dependent Peroxidase

“…Because its gene is located close to genes encoding proteins of the pyruvate dehydrogenase complex, it was annotated as its E3 component. However, LpdA in Streptococcus pneumonia is not associated with any two oxo-acid dehydrogenase complexes (30). In any case, recombinant SucB, PDHB, and LpdA presented disulfide reductase activity ( Fig.…”

“…Comparison of K m values is difficult because the parameters for SucB and PDHB (Table 1) were calculated in a fixed concentration of flavoenzyme (Lpd ϭ 1 M), whereas for LpdA, the concentration of flavoenzyme present in the reaction mixture varies according to the value indicated on the x axis (Fig. 2D) because this enzyme possesses both an Lpd and a lipoyl binding domain (30). To test whether the increase in peroxidase activity could also be influenced by an increase in Lpd concentration, we performed the assay in the presence of SucB and various concentrations of Lpd (supplemental Fig.…”

“…Furthermore, another study detected Ohr and LpdA in the extracellular environment through proteomic analyses of X. fastidiosa (28). LpdA is an enzyme harboring its own substrate, a lipoyl group (30).…”

Role of Dihydrolipoamide Dehydrogenase in Regulation of Raffinose Transport in Streptococcus pneumoniae

“…The production of an expression vector for DLDH has already been described (17). Induction and expression of DLDH was done as described for RafK, with the exception that DLDH was precipitated in 100% ammonium sulfate directly after elution from the Ni-agarose and protein not used immediately was stored at 4°C with little to no loss of enzymatic activity, measured as described in reference 41.…”