Kinetic and crystallographic studies of Escherichia coli UDP‐N‐acetylmuramate:L‐alanine ligase

Abstract

Uridine diphosphate-N-acety1muramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studied included: cation specificity, ionic strength, buffer composition and pH.At 37 "C and pH 8.0, kc, was equal to 980 40 min", while K, values for ATP, UNAM, and L-alanine were, 130 ? 10, 44 ? 3, and 48 -+ 6 wM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity.Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of V,,,/K, for the three substrates and of V,,, were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, fl = 105", and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kc,, for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine.

Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP- N -Acetylmuramic Acid: l -Alanine Ligase (MurC) from Haemophilus influenzae

“…Data collection statistics are presented in Table 1. Neither of these two crystal forms is similar to a previously reported crystal form of E. coli MurC (11). SAD phasing, model building, and refinement.…”

Comparison of the UDP- N -Acetylmuramate: l -Alanine Ligase Enzymes from Mycobacterium tuberculosis and Mycobacterium leprae

“…The properties of these two ligases are very similar. The calculated apparent K m for Gly of both ligases was found to be much lower than the reported K m value of ϳ2.5 to 10 mM for Gly of E. coli MurC (7,12). The mycobacterial MurCs also had similar K m values for L-Ala, and in both cases, this value was slightly lower than the K m for Gly.…”

“…The genes that encode MurC from several organisms have been sequenced (1,6,8,11,13), and the Escherichia coli MurC has been overexpressed and characterized (7,11). However, the mycobacterial counterparts have not been studied.…”

Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP- N -Acetylmuramate:Amino Acid Ligase Activity

“…E. coli MurC possesses the ability to add L-alanine, L-serine, or glycine to UDP-MurNAc (5,11,17). The purified chlamydial MurC also showed ligase activity with radioactive L-alanine, L-serine, or glycine as a substrate, consistent with the formations of UDP-MurNAc-L-Ala, UDP-MurNAc-L-Ser, and UDP-MurNAc-Gly, respectively.…”

“…MurC enzymes from conventional bacteria have the capacity to add L-alanine, L-serine, or glycine to UDP-MurNAc in vitro (5,11,17,20,32). In general, the in vitro and in vivo results correlate well; i.e., the best amino acid substrate in vitro is the one which is added in vivo.…”