Staphylococcus haemolyticus contains two D-glutamic acid biosynthetic activities, a glutamate racemase and a D-amino acid transaminase

Abstract

Two D-glutamic acid biosynthetic activities, glutamate racemase and D-amino acid transaminase, have been described previously for bacteria. To date, no bacterial species has been reported to possess both activities. Genetic complementation studies using Escherichia coli WM335, a D-glutamic acid auxotroph, and cloned chromosomal DNA fragments from Staphylococcus haemolyticus revealed two distinct DNA fragments containing open reading frames which, when present, allowed growth on medium without exogenous D-glutamic acid. Amino acid sequences of the two open reading frames derived from the DNA nucleotide sequences indicated extensive identity with the amino acid sequence of Pediococcus pentosaceous glutamate racemase in one case and with that of the D-amino acid transaminase of Bacillus spp. in the second case. Enzymatic assays of lysates of E. coli WM335 strains containing either the cloned staphylococcal racemase or transaminase verified the identities of these activities. Subsequent DNA hybridization experiments indicated that Staphylococcus aureus, in addition to S. haemolyticus, contained homologous chromosomal DNA for each of these genes. These data suggest that S. haemolyticus, and probably S. aureus, contains genes for two D-glutamic acid biosynthetic activities, a glutamate racemase (dga gene) and a D-amino acid transaminase (dat gene).Most bacteria contain the macromolecular polymer peptidoglycan, which provides tensile strength to the cell wall (13). Peptidoglycan consists of alternating units of N-acetylglucosamine and N-acetylmuramic acid. Alternating L-and Damino acids are added by a set of unique enzymes to UDP-Nacetylmuramic acid to form the final cytoplasmic precursor of peptidoglycan (2, 13). One of the key D-amino acids, D-glutamic acid, has been found in earlier studies to have two different routes of biosynthesis. In Bacillus spp., D-glutamic acid is formed from D-alanine and ␣-ketoglutarate by a D-amino acid transaminase (EC 2.6.1.21) (14,22,24,25), while Lactobacillus spp., Pediococcus spp., and Escherichia coli use racemases which convert L-glutamic acid to D-glutamic acid (E. coli Dga [Glr, MurI]; EC 5.1.1.3) (3-6, 8, 9, 16, 23, 28). Attempts to detect either of these activities biochemically in most other bacteria, including medically important pathogens, have been limited and in some cases unsuccessful (17).Only one D-glutamic acid auxotrophic strain, E. coli WM335, has been reported (12, 15). Attempts to grow WM335 in the absence of exogenous D-glutamic acid result in rapid cell lysis. Using this strain as a recipient to screen a random Staphylococcus haemolyticus library, we observed in the work reported here that S. haemolyticus contains two distinct genes that complement the D-glutamic acid requirement of E. coli WM335. These genes, designated dga and dat, encode glutamate racemase and D-amino acid transaminase enzyme activities, respectively, as determined from in vivo complementation of D-glutamic acid auxotrophy, similarities at the amino acid level to previously reported sequences...

Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis

“…YM-1 and perhaps Staphylococcus aureus have both dat and the racemase gene that can functionally complement the D-glutamate-requirement of an E. coli murI mutant. However, whether both genes are required for D-glutamate synthesis by host cells remains unknown (Fotheringham et al, 1998;Pucci et al, 1995). B. subtilis has yheM, which encodes a putative D-amino acid aminotransferase that is similar (65 % amino acid sequence similarity) to S. haemolyticus Dat aminotransferase (Kunst et al, 1997).…”

Functional Comparison of the Two Bacillus anthracis Glutamate Racemases

“…Therefore, in addition to its proposed role in cell wall biosynthesis, glutamate racemase is also predicted to be the major source of D-glutamate for PDGA capsule synthesis in B. anthracis (9). In contrast to most bacteria that possess only one glutamate racemase gene (5,10,14,18,19,26,29,33,35,40,61), the B. anthracis genome contains two genes (BAS0806 and BAS4379) predicted to encode glutamate racemases, which are designated racE1 and racE2. Recently, inactivation of racE2 was reported to cause a severe growth defect in B. anthracis, while inactivation of racE1 only moderately inhibited growth (52).…”

“…5.1.1.3), which catalyzes the reversible stereoinversion of L-glutamate (5,14,31). Insights into the cofactor-independent amino acid racemases have begun to emerge from biochemical studies of enzymes isolated from several organisms, including Bacillus subtilis, Bacillus pumilus, Bacillus sphaericus, Escherichia coli, Lactobacillus fermentum, Lactobacillus brevis, Aquifex pyrophilus, Staphylococcus haemolyticus, Brevibacterium lactofermentum, and Mycobacterium tuberculosis (1,2,5,10,14,18,19,21,28,29,33,35,40,47,58,61), as well as the recently described crystal structure of RacE-D-glutamate from B. subtilis (43). Several studies have identified glutamate racemase as an essential gene in B. subtilis and E. coli, which has led to the prediction that glutamate racemase activity is important for peptidoglycan biosynthesis in these organisms (14,31).…”

Functional and Structural Characterization of Thermostable d -Amino Acid Aminotransferases from Geobacillus spp

“…D-AAT activity is found in various gram-positive bacteria, including Bacillus (8,28,29), Staphylococcus (22), and Listeria (31), and yet recent biotechnology studies have mainly focused on thermophilic or mesophilic Bacillus enzymes due to their high catalytic activity and broad substrate specificity (4,9,10,23). For example, the D-AAT from thermophilic Bacillus sp.…”