Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we describe three distinct mechanisms of regulation for the family of glutamate racemases: allosteric activation by metabolic precursors, kinetic regulation through substrate inhibition, and D-glutamate recycling using a d-amino acid transaminase. In a search for selective inhibitors, we identified a series of uncompetitive inhibitors specifically targeting Helicobacter pylori glutamate racemase that bind to a cryptic allosteric site, and used these inhibitors to probe the mechanistic and dynamic features of the enzyme. These structural, kinetic and mutational studies provide insight into the physiological regulation of these essential enzymes and provide a basis for designing narrow-spectrum antimicrobial agents.
Formylation of the initiator methionyl-tRNA, catalyzed by methionyl-tRNA formyltransferase, has long been regarded as essential for initiation of protein synthesis in eubacteria. Here, we show that this process is, in fact, dispensable in Pseudomonas aeruginosa. Disruption of the chromosomal methionyl-tRNA formyltransferase gene in P. aeruginosa resulted only in a moderate decrease in the rate of cell growth, whereas in Escherichia coli cell growth was severely impaired. The ability of the P. aeruginosa mutant strain to grow was not due to an additional copy of the methionyl-tRNA formyltransferase gene or to N-acylation of the methionyl moiety by a group other than formyl. These results indicate that P. aeruginosa can carry out formylation-independent initiation of protein synthesis, using the nonformylated methionyl-tRNA. Therefore, the dogma that eubacteria require formylation of the initiator methionyltRNA for initiation of protein synthesis may have been an invalid generalization of results obtained with E. coli.Protein synthesis in eubacteria, and in the chloroplasts and mitochondria of eukaryotes, can be initiated using the initiator formyl-methionyl-tRNA (fMet-tRNA fMet ) 1 (1). Formylation is specific for the initiator methionyl-tRNA (Met-tRNA fMet ) and is catalyzed by methionyl-tRNA formyltransferase (MTF), which is encoded by the fmt gene (2-5). The features in the Escherichia coli tRNA fMet required for formylation are the base-base mismatch between nucleotides 1 and 72 and the second and third base pairs of the acceptor stem (4 -6). The fMet moiety allows initiation factor IF-2 to recognize the initiator tRNA and reject other tRNAs. This conclusion is based on in vivo analysis of the effect of overproduction of IF-2 on the activity of E. coli tRNA fMet mutants defective in formylation (7, 8) and on in vitro studies of IF-2 interaction with N-blocked aminoacyl-tRNA (9, 10). The formyl group also prevents the tRNA fMet from participating in elongation by blocking binding of elongation factor EF-Tu (5, 7). Formylation of the Met-tRNA fMet is generally accepted as a key checkpoint required for initiation of protein synthesis in eubacteria. This dogma, based primarily on studies conducted in E. coli, was further substantiated by the finding that disruption of the E. coli chromosomal fmt gene severely curtailed cell growth (3). However, some earlier studies have obtained circumstantial evidence that questioned this generalization. A Streptococcus faecalis folate-deficient strain, which is unable to synthesize the formyl donor N 10 -formyltetrahydrofolate (fTHF), was shown to be unaffected in growth or viability (11). However, it was not clear that S. faecalis can initiate protein synthesis in the absence of formylation, since the strain also incurred additional chromosomal mutations that affect proper tRNA modification (11).While the studies in S. faecalis were inconclusive, they hinted that formylation may not be a prerequisite for initiation of protein synthesis in all eubacteria. Therefore, we investig...
Formylation of the initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is an essential step in initiation of protein synthesis in eubacteria. Here, site-directed mutagenesis was used to identify active site residues of the Haemophilus influenzae MTF. Of the nine residues investigated, only Arg-41, Asn-107, His-109 and Asp-145 were important for the function of the H. influenzae MTF. Replacement of these residues with Ala resulted in a significant reduction in the efficiency of catalysis. Intrinsic fluorescence analysis indicated that this was not due to a defect in N10-formyltetrahydrofolate (fTHF) binding. The Asp-145 and Arg-41 mutations reduced the affinity of the enzyme for the initiator tRNA, whereas the Asn-107 and His-109 mutations affected catalysis but not tRNA binding. Replacement of Arg-41, His-109 and Asp-145 with functionally similar residues also affected the activity of the enzyme. The data suggest that Asn-107, His-109 and Asp-145 are catalytic residues, whereas Arg-41 is involved in tRNA recognition. In the Escherichia coli glycinamide ribonucleotide formyltransferase, which also uses fTHF as the formyl donor, Asn-106, His-108 and Asp-144 participate in the catalytic step. Together, these observations imply that this group of enzymes uses the same basic mechanism in formylating their substrates.
Formylation of the initiator methionyl-tRNA (Met-tRNA fMet ) in eubacteria is catalyzed by methionyl-tRNA formyltransferase (MTF). Features of the Escherichia coli tRNA fMet that are important for formylation are the base-base mismatch between nucleotides 1 and 72, and the second and third base pairs of the acceptor stem. The base-base mismatch is the most crucial formylation determinant in the E. coli tRNA fMet . However, it is not known whether this feature is also important for formylation of other eubacterial tRNA fMet . We cloned the Pseudomonas aeruginosa MTF gene by complementation of an E. coli MTF mutant strain with a genomic library, and investigated the catalytic properties and substrate specificity of the enzyme. The results show that the P. aeruginosa and E. coli enzymes have comparable affinities for the tRNA fMet and N 10 -formyltetrahydrofolate (fTHF) substrates. Overproduction of the P. aeruginosa MTF rescued the initiator activity of an E. coli formylation-defective tRNA fMet with a base pair between nucleotides 1 and 72, indicating that the base-base mismatch is utilized by the P. aeruginosa MTF for recognition of the tRNA fMet . Therefore, this feature may be used by MTFs from other eubacteria to distinguish the initiator from elongator tRNAs. ß
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