Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) catalyzes the ring cleavage step in the catabolism of aromatic compounds through the protocatechuate branch of the -ketoadipate pathway. A protocatechuate 3,4-dioxygenase was purified from Streptomyces sp. strain 2065 grown in p-hydroxybenzoate, and the N-terminal sequences of the -and ␣-subunits were obtained. PCR amplification was used for the cloning of the corresponding genes, and DNA sequencing of the flanking regions showed that the pcaGH genes belonged to a 6.5-kb protocatechuate catabolic gene cluster; at least seven genes in the order pcaIJFHGBL appear to be transcribed unidirectionally. Analysis of the cluster revealed the presence of a pcaL homologue which encodes a fused ␥-carboxymuconolactone decarboxylase/-ketoadipate enol-lactone hydrolase previously identified in the pca gene cluster from Rhodococcus opacus 1CP. The pcaIJ genes encoded proteins with a striking similarity to succinyl-coenzyme A (CoA):3-oxoacid CoA transferases of eukaryotes and contained an indel which is strikingly similar between high-G؉C gram-positive bacteria and eukaryotes.The intradiol or ortho ring cleavage pathway commonly known as the -ketoadipate pathway, named after the intermediate -ketoadipate (3-oxoadipate) (57), is widely distributed among taxonomically diverse soil microorganisms, including both eubacteria and fungi. It is considered a "major utility pathway" playing a significant role in the processing and degradation of aromatic compounds from plant material found in soil, such as those originating from the solubilization of lignin (27). The pathway consists of two branches, one starting at catechol and the other at protocatechuic acid, which are cleaved by catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase (3,4-PCD), respectively. In bacteria the two branches of the pathway converge at the intermediate -ketoadipate enol-lactone. The -ketoadipate pathway is biochemically conserved, and the structural genes encoding enzymes of this pathway ( Fig. 1) in widely differing bacterial species are similar. The genes of the -ketoadipate pathway have been cloned and sequenced from different bacteria, including Acinetobacter sp. strain ADP1 and Pseudomonas putida, two organisms whose GϩC contents differ by 20%, but amino acid sequence identities for isofunctional Pca enzymes range from 45 to 68% (27). Despite this conservation, diversity in the -ketoadipate pathways has evolved in pathway branching, inducing metabolites, genetic organization, operon clustering, and regulation (48).In addition to its role in the degradation of aromatic compounds derived from lignin and other plant compounds that are recalcitrant or resistant to degradation, the enzymes of the -ketoadipate pathway are required for the degradation of chlorocatechols by the modified ortho pathway. Although a 3,4-dihydroxychlorobenzoic acid ortho-cleaving enzyme has not been found, two protocatechuate 3,4-dioxygenase isozymes that oxidize 4-sulfocatechol were identified recently from two members ...
A gene encoding high potential iron sulfur protein (HiPIP) iso-1 from Ectothiorhodospira halophila was constructed in one step from long synthetic oligonucleotides. The gene was inserted into a phagemid vector from which the HiPIP was expressed as a fusion protein to > 10% of the soluble protein in Escherichia coli, demonstrating that a 4Fe-4S protein can be highly expressed in E. coli. The recombinant HiPIP was purified to apparent homogeneity by affinity chromatography followed by proteolytic removal of the leader sequence and anion exchange chromatography. Approximately 180 mg of HiPIP were purified from 10 l of cell culture. CD spectra of the oxidized and reduced forms of the protein and the 1H NMR spectrum of the oxidized protein are essentially identical to those of the wild type protein, indicating that the environment of the iron sulfur cluster in the two proteins is the same and thus that the recombinant protein is folded correctly. The reduction potential of the recombinant protein was determined to be 120 +/- 6 mV versus NHE (20 mM HEPES, 0.1 M sodium chloride, pH 7.0, 25 degrees C). This efficient heterologous expression of an HiPIP enables a systematic investigation of structure-function relationships in this class of iron sulfur proteins.
Conserved tyrosine-12 of Ectofhiorhodospira hdophikz high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y 12H), tryptophan (Y 12W), isoleucine (Y 12I), and alanine (Y 12A). Variants Y 12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y 12F, Y12H, and Y 12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-1 1 .O. Characterization of the Y 12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 0 6 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-591 Val-58 peptide bond, thereby perturbing Gly-46. The AAG;j.. of Y12F of 2.3 kcal/mol with respect to rcWT HiPlP (25 "C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y 12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 "C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.
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