A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na ؉ -translocating F 1 F 0 ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na ؉ ions but not in its presence, indicating the specific reaction with the Na ؉ binding c-Glu 65 residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu 65 was therefore in close contact with phospholipid but does not contact subunit a. Na ؉ in / 22 Na ؉ out exchange activity of the ATP synthase was not affected by modifying the c-Glu 65 sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na ؉ ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospolipases C and A 2 and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu 65 is located within the core of the membrane.

UDP-N-acetylmuramyl:L-alanine ligase from Escherichia coli was overexpressed more than 600-fold and purified to near homogeneity. The purified enzyme was found to ligate L-alanine, L-serine, and glycine, as well as the nonnatural amino acid ␤-chloro-L-alanine, to UDP-N-acetylmuramic acid. On the basis of (i) the specificity constants of the enzyme determined for L-alanine, L-serine, and glycine and (ii) the levels of these amino acids in the intracellular pool, it was calculated that the rates of incorporation of L-serine and glycine into peptidoglycan precursor metabolites could maximally amount to 0.1 and 0.5%, respectively, of the rate of L-alanine incorporation.The pathway for the biosynthesis of peptidoglycan precursor metabolites involves (i) reactions for the synthesis of UDPactivated amino sugars and (ii) reactions for the synthesis of the peptide moiety of UDP-N-acetylmuramyl-pentapeptide (2). Four nonribosomal amino acid ligases catalyze the sequential addition of L-alanine, D-glutamate, meso-diaminopimelic acid (mDAP) or L-lysine (in gram-positive cocci), and D-alanyl-D-alanine dipeptide to UDP-N-acetylmuramic acid (UDPMurNAc). The amino acid-adding enzymes have been characterized in cell extracts or in partially purified form from a wide variety of bacterial species (3,7,10,(15)(16)(17)(18)20). Their ubiquitous occurrence indicates that all bacteria synthesizing peptidoglycan cell walls possess this set of four amino acid-adding enzymes.This work reports the overexpression, purification, and substrate specificity of UDP-MurNAc:L-alanine ligase from Escherichia coli. Similar work on the same enzyme was published (14) while this paper was being written.UDP-MurNAc:L-alanine ligase catalyzes the addition of the first amino acid to the lactyl group of UDP-MurNAc as follows: UDP-MurNAc ϩ L-alanine ϩ ATP3UDP-MurNAc-Lalanine ϩ ADP ϩ P i . The excellent stability of the enzyme preparation and its high specific activity, in conjunction with the use of a coupled spectrophotometric assay, allowed the determination of the kinetic constants K m and V max of the L-alanine-adding enzyme in the presence of different amino acid substrates. Investigation of the kinetic properties of the enzyme elucidated why biosynthesis of peptidoglycan precursor metabolites in E. coli is highly specific in spite of the fact that UDP-MurNAc:L-alanine ligase accepts L-serine and glycine, besides L-alanine, as substrates.(This research was conducted by Yvonne Appoldt in partial fulfillment of the requirements for a M.S. from the University of Basel, Basel, Switzerland, 1995.) Overexpression and purification of UDP-MurNAc:L-alanine ligase. The murC gene from E. coli, which codes for UDPMurNAc:L-alanine ligase, was amplified by PCR and cloned in

The atpB and atpF genes of Propionigenium modestum were cloned as His-tag fusion constructs and expressed in Escherichia coli. Both recombinant subunits a and b were purified via Ni 2+ chelate affinity chromatography. A functionally active F o complex was reassembled in vitro from subunits a, b and c, and incorporated into liposomes. The F o liposomes catalysed 22 Na + uptake in response to an inside negative potassium diffusion potential, and the uptake was prevented by modification of the c subunits with N,N¢-dicyclohexylcarbodiimide (DCCD). In the absence of a membrane potential the F o complexes catalysed 22 Na + out / Na + in -exchange. After F 1 addition the F 1 F o complex was formed and the holoenzyme catalysed ATP synthesis, ATP dependent Na + pumping, and ATP hydrolysis, which was inhibited by DCCD. Functional F o hybrids were reconstituted with recombinant subunits a and b from P. modestum and c 11 from Ilyobacter tartaricus. These F o hybrids had Na + translocation activities that were not distinguishable from that of P. modestum F o .

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