Muconate-lactonizing enzyme

Figure 4.9 Mechanisms of the reactions catalyzed by the enzymes mandelate racemase (a) and muconate lactonizing enzyme (b). The two overall reactions are quite different a change of configuration of a carbon atom for mandelate racemase versus ring closure for the lactonizing enzyme. However, one crucial step (red) in the two reactions is the same addition of a proton (blue) to an intermediate of the substrate (red) from a lysine residue of the enzyme (E) or. In the reverse direction, formation of an intermediate by proton abstraction from the carbon atom adjacent to the carboxylate group.

Goldman, A., Ollis, D.L., Steitz, T.A. Crystal structure of muconate lactonizing enzyme at 3 A resolution. 

Neidhart, D.J., et al. Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous. Nature 347 ... 

Another enzyme of the mandelate pathway of degradation of aromatic rings (Fig. 25-8) is the cis,cis-muconate lactonizing enzyme which catalyzes the reaction of Eq. 13-23. It has a three-dimensional struc-... 

The mandelate and jS-ketoadipate pathways serve as an example of gene duplication, as there is strong evidence pointing to the former evolving from the latter. Evolution of mandelate racemase from muconate lactonizing enzyme points to the relevance of the enzyme mechanism for catalytic reactivity. 

Enolases mandelate racemase (MR), muconate-lactonizing enzyme (MLE), N-acetylamino acid racemase (NAAR) Hasson, 1998 Palmer, 1999... 

MR, an a/f-barrel protein, features a high degree of similarity with muconate-lactonizing enzyme (MLE) of the /3-ketoadipate metabolism 26% sequence identity, the same symmetry and organization of subunits (octamer), a divalent metal ion requirement (Mn2+ for MLE, Mg + for MR). As MLE is almost ubiquitous among Pseudomonas, compared to 5% frequency of MR, it can be concluded that MLE is the older enzyme and probably the precedessor of MR. 

P. C. Babbitt, J. A. Gerlt, G. A. Petsko, and D. Ringe, Evolution of an enzyme active site the structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase, Proc. Natl. Acad. Sci. USA 1998,... 

The enolase superfamily story started with the serendipitous discovery that two enzymes catalyzing very different overall reactions, mandelate racemase (MR) and muconate lactonizing enzyme (MLE), had virtually superimposable structures (Neidhart et al., 1990). As shown in Figure 2, MR catalyzes the reversible racemization of mandelate, an aromatic substrate, while MLE catalyzes the equilibration of muconolactone with as, m-muconate. Given the substantial differences in these reactions, it... 

Isomerases catalyze conversions within one molecule. For example, the cis.cis-muconate lactonizing enzyme (cycloisomerase) catalyzes the chiral conversion of ds,cis-muconic acid to (K)-muconolactone (Fig. 29). It is a key enz)one in the degradation of benzoate via the p-ketoadipate pathway (109). Chiral lactones could be useful as chiral synthons. 

Figure 8 X-ray diffraction images, (a) A precession photograph of muconate lactonizing enzyme. The fourfold symmetry in the diffraction pattern is clearly visible. This gives an undistorted view of the reciprocal lattice but are no longer used because they are not as efficient as rotation images, (b) A rotation image of hen s egg white lysozyme. This easily obtainable image gives a distorted projection of the reciprocal lattice, but this is no obstacle for modern programs.

Ransom et al. 193 cloned the gene for mandelate racemase from Pseudomonas putida in Pseudomonas aeruginosa on the basis of the inability of the latter strain to grow on D-mandelate as a sole carbon source. The amino acid sequence was deduced from the nucleotide sequence, and the predicted molecular mass of the enzyme was 38 750[193. The enzyme is composed of eight identical subunits. The crystal structure of mandelate racemase has been solved and refined at 2.5 A resolution [194. The secondary, tertiary and quaternary structures of mandelate racemase are quite similar to those of muconate lactonizing enzyme 195, 196. Mandelate racemase is composed of two major structural domains and a small C-terminal domain. The N-terminal domain has an a + P structure, and the central domain has an a/p-barrel topology. The C-terminal domain consists of an L-shaped loop. 

Other cases in which the native and promiscuous activities differ in the 1st digit include muconate lactonizing enzyme (MLE Table 1, entry 9), whose native activity is cycloisomerization (EC 5.5.1.1) and possesses a promiscuous OSBS (/3-elimination) activity (EC 4.2.1.113). 

The X-ray crystal structure of P. putida muconate lactonizing enzyme (cycloisomerase) was determined in 1987, and was found to contain an a/(i barrel fold, also found in triosephosphate isomerase and enolase. Remarkably, the structure of P. putida mandelate racemase, which catalyzes a mechanistically distinct reaction earlier in the same pathway, was found in 1990 to have a homologous structure, indicating that the structural fold of the enolase superfamily is able to support a range of enzyme-catalyzed reactions. The P. putida 3-carboxy- r, x-muconate lactonizing enzyme, in contrast, shares sequence similarity with a class II fumarase enzyme, and determination of its structure in 2004 has shown that it shares the same fold as the class II fumarase superfamily, hence these two catalysts of similar reactions have evolved from different ancestors. ... 

E. coli = Escherichia coli ET = evolutionary trace analysis MLE = muconate lactonizing enzyme MR = mandelate racemase ORF = open reading frame PAM = number of accepted point mutations per 100 residues separating two sequences PEP = phosphoenolpyruvate. 

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