Q ) rms(∆δ meas -∆δ pred )/rms(∆δ meas )(3) Communications to the Editor

Fosfomycin [(1R,2S)-1,2-epoxypropylphosphonic acid] has been shown to exert its antibiotic effect through the inhibition of UDP-GlcNAc enolpyruvoyl transferase [Kahan, F. M., et al. (1974) Ann. N.Y. Acad. Sci. 235, 364], the enzyme responsible for catalyzing the first committed step in bacterial cell wall biosynthesis. Time-dependent inactivation of MurZ by fosfomycin was found to be greatly accelerated by the presence of cosubstrate UDP-GlcNAc but could also be speeded appreciably by the unreactive substrate analog 3-deoxy-UDP-GlcNAc. These results argue against a reaction-based participation of the cosubstrate and suggest that UDP-GlcNAc has a role in influencing active site conformation critical to the inactivation event. A study of the influence of UDP-GlcNAc and fosfomycin on the kinetics of inactivation allowed the determination of dissociation constants for fosfomycin (KF = 8.6 microM) and UDP-GlcNAc (KS = 14 microM), in addition to a limiting inactivation rate constant (k(inact) = 7.4 min-1) at saturating UDP-GlcNAc and fosfomycin concentrations. Mass spectrometry of inactivated MurZ demonstrated an increase in molecular weight of 138, consistent with the covalent addition of a molar equivalent of fosfomycin (136 kDa). Titration of MurZ with fosfomycin revealed a stoichiometry of 1 molecule of inhibitor per active site when assessed using either enzyme activity or mass spectrometry as an index of modification. Peptide mapping of tryptic digests of fosfomycin-inactivated MurZ revealed modification of a unique 41-mer, the sequence of which revealed that Cys115 was the site of attachment of fosfomycin.

The Escherichia coli gene murZ, encoding the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase, has been cloned and sequenced. Identified by screening an E. coli genomic library for clones that conferred phosphomycin resistance, murZ encoded a 419-amino-acid polypeptide and was mapped to 69.3 min on the E. coli chromosome. MurZ protein was purified to near homogeneity and found to have the expected UDP-N-acetylglucosamine enolpyruvyl transferase activity. Sequence analysis of the predicted product revealed 44% identity to OrfR from BaciUlus subtilis (K. Trach, J.

The recently isolated Escherichia coli murB gene (Pucci et al., 1992) has been cloned into an expression vector and the encoded UDP-N-acetylenolpyruvylglucosamine reductase (EC 1.1.1.158) was overproduced to about 10% of soluble cell protein. The encoded 38-kDa protein has been purified to near homogeneity. It was found to be a monomer and to contain stoichiometric amounts of bound FAD which is reducible in catalytic turnover. The enzyme utilizes the 4-pro-S hydrogen of NADPH to reduce the enolpyruvyl group of UDP-N-acetylglucosamine enolpyruvate to the lactyl ether in UDP-N-acetylmuramic acid. NMR analysis of products from 2H2O and 4S-[2H]NADPH incubations establishes that a hydride from NADPH via E.FADH2 is transferred to the beta-methyl of the 3-O-lactyl moiety and a proton from solvent to the alpha-carbon of the lactyl moiety of UDP-N-acetylmuramic acid. A mechanism for this unusual enolether reduction in bacterial cell wall assembly is proposed.