Crystal structure of RimI from Salmonella typhimurium LT2, the GNAT responsible for N(alpha)-acetylation of ribosomal protein S18.

The three ribosomal proteins L7, S5, and S18 are included in the rare subset of prokaryotic proteins that are known to be N(alpha)-acetylated. The GCN5-related N-acetyltransferase (GNAT) protein RimI, responsible for the N(alpha)-acetylation of the ribosomal protein S18, was cloned from Salmonella typhimurium LT2 (RimI(ST)), overexpressed, and purified to homogeneity. Steady-state kinetic parameters for RimI(ST) were determined for AcCoA and a peptide substrate consisting of the first six amino acids of the target protein S18. The crystal structure of RimI(ST) was determined in complex with CoA, AcCoA, and a CoA-S-acetyl-ARYFRR bisubstrate inhibitor. The structures are consistent with a direct nucleophilic addition-elimination mechanism with Glu103 and Tyr115 acting as the catalytic base and acid, respectively. The RimI(ST)-bisubstrate complex suggests that several residues change conformation upon interacting with the N terminus of S18, including Glu103, the proposed active site base, facilitating proton exchange and catalysis.

TetX is a flavin-dependent monooxygenase conferring resistance to tetracycline antibiotics.

The tetracycline antibiotics block microbial translation and constitute an important group of antimicrobial agents that find broad clinical utility. Resistance to this class of antibiotics is primarily the result of active efflux or ribosomal protection; however, a novel mechanism of resistance has been reported to be oxygen-dependent destruction of the drugs catalyzed by the enzyme TetX. Paradoxically, the tetX genes have been identified on transposable elements found in anaerobic bacteria of the genus Bacteroides. Overexpression of recombinant TetX in Escherichia coli followed by protein purification revealed a stoichiometric complex with flavin adenine dinucleotide. Reconstitution of in vitro enzyme activity demonstrated a broad tetracycline antibiotic spectrum and a requirement for molecular oxygen and NADPH in antibiotic degradation. The tetracycline products of TetX activity were unstable at neutral pH, but mass spectral and NMR characterization under acidic conditions supported initial monohydroxylation at position 11a followed by intramolecular cyclization and non-enzymatic breakdown to other undefined products. TetX is therefore a FAD-dependent monooxygenase. The enzyme not only catalyzed efficient degradation of a broad range of tetracycline analogues but also conferred resistance to these antibiotics in vivo. This is the first molecular characterization of an antibiotic-inactivating monooxygenase, the origins of which may lie in environmental bacteria.

Functionally important amino acids in Saccharomyces cerevisiae aspartate kinase.

Saccharomyces cerevisiae aspartate kinase (AK(Sc)) phosphorylates L-Asp as the first step in the aspartate pathway responsible for the biosynthesis of L-Thr, L-Met, and L-Ile in microorganisms and plants. Using site-directed mutagenesis, we have evaluated the importance of residues in AK(Sc) that are strongly conserved among aspartate kinases or in other small molecule kinases. Steady state kinetic analysis of the purified AK(Sc) variants reveals that several of the targeted amino acids, particularly K18 and H292, have important roles in the enzymatic reaction. These results provide the first identification of amino acid residues crucial to the action of this important metabolic enzyme.