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Query: EC:5.5.1.1 (muconate lactonizing enzyme)
 85 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter "calcoaceticus" ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis, cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.
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PMID:Substrate specificity of and product formation by muconate cycloisomerases: an analysis of wild-type enzymes and engineered variants. 972 73

The structure of (D)-glucarate dehydratase from Pseudomonas putida (GlucD) has been solved at 2.3 A resolution by multiple isomorphous replacement and refined to a final R-factor of 19.0%. The protein crystallizes in the space group I222 with one subunit in the asymmetric unit. The unit cell dimensions are a = 69.6 A, b = 108.8 A, and c = 122.6 A. The crystals were grown using the batch method where the primary precipitant was poly(ethylene glycol) 1000. The structure reveals that GlucD is a tetramer of four identical polypeptides, each containing 451 residues. The structure was determined without a bound substrate or substrate analogue. Three disordered regions are noted: the N-terminus through residue 11, a loop containing residues 99 through 110, and the C-terminus from residue 423. On the basis of primary sequence alignments, we previously concluded that GlucD is a member of the mandelate racemase (MR) subfamily of the enolase superfamily [Babbitt, P. C., Hasson, M. S., Wedekind, J. E., Palmer, D. R. J., Barrett, W. C., Reed, G. J., Rayment, I., Ringe, D., Kenyon, G. L., and Gerlt, J. A. (1996) Biochemistry 35, 16489-16501]. This prediction is now verified, since the overall fold of GlucD is strikingly similar to those of MR, muconate lactonizing enzyme I, and enolase. Also, many of the active site residues of GlucD can be superimposed on those found in the active site of MR. The implications of this structure on the evolution of catalysis in the enolase superfamily are discussed.
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PMID:Evolution of enzymatic activities in the enolase superfamily: crystal structure of (D)-glucarate dehydratase from Pseudomonas putida. 977 61

We have refined to 2.3 A resolution two muconate cycloisomerase (MCIase) variant structures, F329I and I54V, that differ from each other and from wild-type in their activity toward cis,cis-muconate (CCM) and substituted CCMs. The working and free R-factors for F329I are 17.4/21.6% and for I54V, 17.6/22.3% with good stereochemistry. Except for the mutated residue, there are no significant changes in structure. To understand the differences in enzymatic properties we docked substituted CCMs and CCM into the active sites of the variants and wild type. The extra space the mutations create appears to account for most of the enzymatic differences. The lack of other structural changes explains why, although structurally equivalent changes occur in chloromuconate cycloisomerase (CMCIase), the changes in themselves do not convert a MCIase into a dehalogenating CMCIase. Reanalysis of the CMCIase structure revealed only one general acid/base, K169. The structural implication is that, in 2-chloro-CCM conversion by CMCIase, the lactone ring of 5-chloromuconolactone rotates before dehalogenation to bring the acidic C4 proton next to K169. Therefore, K169 alone performs both required protonation and deprotonation steps, the first at C5 as in MCIase, and the second, after ring rotation, at C4. This distinguishes CMCIase from alpha/beta barrel isomerases and racemases, which use two different bases.
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PMID:Structural basis for the activity of two muconate cycloisomerase variants toward substituted muconates. 1033 78

A positive selection method for mutations affecting bioconversion of aromatic compounds was applied to a mutant strain of Agrobacterium tumefaciens A348. The nucleotide sequence of the A348 pcaHGB genes, which encode protocatechuate 3,4-dioxygenase (PcaHG) and beta-carboxy-cis,cis-muconate cycloisomerase (PcaB) for the first two steps in catabolism of the diphenolic protocatechuate, was determined. An omega element was introduced into the pcaB gene of A348, creating strain ADO2077. In the presence of phenolic compounds that can serve as carbon sources, growth of ADO2077 is inhibited due to accumulation of the tricarboxylate intermediate. The toxic effect, previously described for Acinetobacter sp., affords a powerful selection for suppressor mutations in genes required for upstream catabolic steps. By monitoring loss of the marker in pcaB, it was possible to determine that the formation of deletions was minimal compared to results obtained with Acinetobacter sp. Thus, the tricarboxylic acid trick in and of itself does not appear to select for large deletion mutations. The power of the selection was demonstrated by targeting the pcaHG genes of A. tumefaciens for spontaneous mutation. Sixteen strains carrying putative second-site mutations in pcaH or -G were subjected to sequence analysis. All single-site events, their mutations revealed no particular bias toward multibase deletions or unusual patterns: five (-1) frameshifts, one (+1) frameshift, one tandem duplication of 88 bp, one deletion of 92 bp, one nonsense mutation, and seven missense mutations. PcaHG is considered to be the prototypical ferric intradiol dioxygenase. The missense mutations served to corroborate the significance of active site amino acid residues deduced from crystal structures of PcaHG from Pseudomonas putida and Acinetobacter sp. as well as of residues in other parts of the enzyme.
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PMID:Positive selection for mutations affecting bioconversion of aromatic compounds in Agrobacterium tumefaciens: analysis of spontaneous mutations in the protocatechuate 3,4-dioxygenase gene. 1102 36

Mutants of the bacterium Acinetobacter sp. strain ADP1 were selected to grow on benzoate without the BenM transcriptional activator. In the wild type, BenM responds to benzoate and cis,cis-muconate to activate expression of the benABCDE operon, which is involved in benzoate catabolism. This operon encodes enzymes that convert benzoate to catechol, a compound subsequently degraded by cat gene-encoded enzymes. In this report, four spontaneous mutants were found to carry catB mutations that enabled BenM-independent growth on benzoate. catB encodes muconate cycloisomerase, an enzyme required for benzoate catabolism. Its substrate, cis,cis-muconate, is enzymatically produced from catechol by the catA-encoded catechol 1,2-dioxygenase. Muconate cycloisomerase was purified to homogeneity from the wild type and the catB mutants. Each purified enzyme was active, although there were differences in the catalytic properties of the wild type and variant muconate cycloisomerases. Strains with a chromosomal benA::lacZ transcriptional fusion were constructed and used to investigate how catB mutations affect growth on benzoate. All of the catB mutations increased cis,cis-muconate-activated ben gene expression in strains lacking BenM. A model is presented in which the catB mutations reduce muconate cycloisomerase activity during growth on benzoate, thereby increasing intracellular cis, cis-muconate concentrations. This, in turn, may allow CatM, an activator similar to BenM in sequence and function, to activate ben gene transcription. CatM normally responds to cis,cis-muconate to activate cat gene expression. Consistent with this model, muconate cylcoisomerase specific activities in cell extracts of benzoate-grown catB mutants were low relative to that of the wild type. Moreover, the catechol 1,2-dioxygenase activities of the mutants were elevated, which may result from CatM responding to the altered intracellular levels of cis,cis-muconate and increasing catA expression. Collectively, these results support the important role of metabolite concentrations in controlling benzoate degradation via a complex transcriptional regulatory circuit.
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PMID:Mutations in catB, the gene encoding muconate cycloisomerase, activate transcription of the distal ben genes and contribute to a complex regulatory circuit in Acinetobacter sp. strain ADP1. 1109 67

Chloromuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-cis,cis-muconate to cis-dienelactone (cis-4-carboxymethylenebut-2-en-4-olide), while usual muconate cycloisomerases transform the same substrate to the bacteriotoxic protoanemonin. Formation of protoanemonin requires that the cycloisomerization of 3-chloro-cis,cis-muconate to 4-chloromuconolactone is completed by protonation of the exocyclic carbon of the presumed enol/enolate intermediate before chloride elimination and decarboxylation take place to yield the final product. The formation of cis-dienelactone, in contrast, could occur either by dehydrohalogenation of 4-chloromuconolactone or, more directly, by chloride elimination from the enol/enolate intermediate. To reach a better understanding of the mechanisms of chloride elimination, the proton-donating Lys169 of Pseudomonas putida muconate cycloisomerase was changed to alanine. As expected, substrates requiring protonation, such as cis,cis-muconate as well as 2- and 3-methyl-, 3-fluoro-, and 2-chloro-cis,cis-muconate, were not converted at a significant rate by the K169A variant. However, the variant was still active with 3-chloro- and 2,4-dichloro-cis,cis-muconate. Interestingly, cis-dienelactone and 2-chloro-cis-dienelactone were formed as products, whereas the wild-type enzyme forms protoanemonin and the not previously isolated 2-chloroprotoanemonin, respectively. Thus, the chloromuconate cycloisomerases may avoid (chloro-)protoanemonin formation by increasing the rate of chloride abstraction from the enol/enolate intermediate compared to that of proton addition to it.
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PMID:Mechanism of chloride elimination from 3-chloro- and 2,4-dichloro-cis,cis-muconate: new insight obtained from analysis of muconate cycloisomerase variant CatB-K169A. 1144 90

Muconate cycloisomerase (MCI) was purified from Rhodococcus rhodochrous 89 grown on phenol. The enzyme appears to contain two different type subunits with molecular masses 35.5 and 37 kD. The N-terminal amino acid sequence of both subunits showed more similarity to corresponding enzymes from gram-negative bacteria than to one from Rhodococcus opacus 1CP. MCI from R. rhodochrous 89, like analogous enzymes from gram-negative bacteria, can convert 2-chloromuconate (2-CM) with the formation of both, 2- and 5-chloromuconolactones (CML) as intermediates. Nevertheless, its unique ability to convert 5-CML to cis- but not to trans-dienelactone sets it apart from all known chloromuconate cycloisomerases from gram-negative and gram-positive bacteria.
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PMID:A new type of muconate cycloisomerase from Rhodococcus rhodochrous strain 89. 1156 54

The members of the mechanistically diverse enolase superfamily catalyze different overall reactions by using a common catalytic strategy and structural scaffold. In the muconate lactonizing enzyme (MLE) subgroup of the superfamily, abstraction of a proton adjacent to a carboxylate group initiates reactions, including cycloisomerization (MLE), dehydration [o-succinylbenzoate synthase (OSBS)], and 1,1-proton transfer (catalyzed by an OSBS that also catalyzes a promiscuous N-acylamino acid racemase reaction). The realization that a member of the MLE subgroup could catalyze a 1,1-proton transfer reaction, albeit poorly, led to a search for other enzymes which might catalyze a 1,1-proton transfer as their physiological reaction. YcjG from Escherichia coli and YkfB from Bacillus subtilis, proteins of previously unknown function, were discovered to be L-Ala-D/L-Glu epimerases, although they also catalyze the epimerization of other dipeptides. The values of k(cat)/K(M) for L-Ala-D/L-Glu for both proteins are approximately 10(4) M(-1) s(-1). The genomic context and the substrate specificity of both YcjG and YkfB suggest roles in the metabolism of the murein peptide, of which L-Ala-D-Glu is a component. Homologues possessing L-Ala-D/L-Glu epimerase activity have been identified in at least two other organisms.
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PMID:Evolution of enzymatic activities in the enolase superfamily: functional assignment of unknown proteins in Bacillus subtilis and Escherichia coli as L-Ala-D/L-Glu epimerases. 1174 47

The members of the enolase superfamily catalyze different overall reactions, yet share a partial reaction that involves Mg(2+)-assisted enolization of the substrate carboxylate anion. The fate of the resulting enolate intermediate is determined by the active site of each enzyme. Several members of this superfamily have been structurally characterized to permit an understanding of the evolutionary strategy for using a common structural motif to catalyze different overall reactions. In the preceding paper, two new members of the superfamily were identified that catalyze the epimerization of the glutamate residue in L-Ala-D/L-Glu. These enzymes belong to the muconate lactonizing enzyme subgroup of the enolase superfamily, and their sequences are only 31% identical. The structure of YcjG, the epimerase from Escherichia coli, was determined by MAD phasing using both the SeMet-labeled protein and a heavy atom derivative. The structure of YkfB, the epimerase from Bacillus subtilis, was determined by molecular replacement using the muconate lactonizing enzyme as a search model. In this paper, we report the three-dimensional structures of these enzymes and compare them to the structure of o-succinylbenzoate synthase, another member of the muconate lactonizing enzyme subgroup.
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PMID:Evolution of enzymatic activities in the enolase superfamily: crystal structures of the L-Ala-D/L-Glu epimerases from Escherichia coli and Bacillus subtilis. 1174 48

We isolated the two LysR-type regulatory proteins CatR1 and CatR2, which regulate the expression of cat1 and cat2 gene clusters, respectively, required for catechol degradation in the bacterium Frateuria sp. ANA-18. In a gel mobility shift assay using CatR1 and the DNA fragment containing the catB1 promoter region, the formation of two complexes, complex 1-1 (C1-1) and complex 1-2 (C1-2), was observed in the presence of cis,cis-muconate. On the other hand, CatR2 and the DNA fragment containing the catB2 promoter region formed only complex 2-2 (C2-2) at a lower concentration of cis,cis-muconate than that at which C1-1 and C1-2 were formed. As the concentration of cis,cis-muconate decreased, the production of the muconate cycloisomerase isozyme MC II encoded by catB2 decreased as well as that of MC I encoded by catB1. However, the amount of MC II synthesized was larger than that of MC I at low concentrations. On the basis of these results, we concluded that the catB2 promoter was activated at low concentrations of cis,cis-muconate.
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PMID:Regulation by two CatR proteins that differ in binding affinity to catB promoters expressing two cat gene clusters. 1175 2


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