Understanding enzyme superfamilies. Chemistry As the fundamental determinant in the evolution of new catalytic activities.
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Animals↗
Biomedical subjects
P C Babbitt
Publications and source records attributed to P C Babbitt.
Cloning and functional characterization of a rat renal organic cation transporter isoform (rOCT1A).
Polyspecific organic cation transporters in the renal proximal tubule mediate the secretion of many clinically used drugs as well as endogenous metabolites. Recently, two organic cation transporters (rOCT1 and rOCT2) were cloned from rat kidney. In this study, we report the cloning and functional expression of an rOCT1 isoform, rOCT1A, from rat kidney. Genomic DNA cloning and sequencing demonstrated that rOCT1A is an alternatively spliced variant of rOCT1 with a deletion of 104 base pairs near the 5'-end. The uptake of [14C]tetraethylammonium (TEA) in oocytes injected with the cRNA-encoding rOCT1A was increased 16-fold over that in water-injected oocytes (29 +/- 2.8 pmol/oocyte/h versus 1.8 +/- 0.13 pmol/oocyte/h, mean +/- S.E., p < 0.05). [14C]TEA uptake in the cRNA-injected oocytes was saturable (Km = 42 +/- 11 microM) and was inhibited significantly by organic cations, including cimetidine and N1-methylnicotinamide. The amino acid sequence was deduced from the cDNA after examination of all three reading frames. Two overlapping open reading frames were found. Studies with synthetic constructs suggest that a functional organic cation transporter is encoded by the larger open reading frame. The larger open reading frame encodes a 430-amino acid protein (termed rOCT1A) that is 92% identical to rOCT1 and 57% identical to rOCT2. From hydropathy analysis, rOCT1A is predicted to have 10 transmembrane domains with both amino and carboxyl termini intracellular. RNase protection assays demonstrate the presence of rOCT1A mRNA transcripts in rat kidney cortex, medulla, and intestine. These studies demonstrate the presence of a functional, alternatively spliced organic cation transporter (rOCT1A) in rat kidney.
Amino Acid Sequence↗
The enolase superfamily: a general strategy for enzyme-catalyzed abstraction of the alpha-protons of carboxylic acids.
We have discovered a superfamily of enzymes related by their ability to catalyze the abstraction of the alpha-proton of a carboxylic acid to form an enolic intermediate. Although each reaction catalyzed by these enzymes is initiated by this common step, their overall reactions (including racemization, beta-elimination of water, beta-elimination of ammonia, and cycloisomerization) as well as the stereochemical consequences (syn vs anti) of the beta-elimination reactions are diverse. Analysis of sequence and structural similarities among these proteins suggests that all of their chemical reactions are mediated by a common active site architecture modified through evolution to allow the enolic intermediates to partition to different products in their respective active sites via different overall mechanisms. All of these enzymes retain the ability to catalyze the thermodynamically difficult step of proton abstraction. These homologous proteins, designated the "enolase superfamily", include enolase as well as more metabolically specialized enzymes: mandelate racemase, galactonate dehydratase, glucarate dehydratase, muconate-lactonizing enzymes, N-acylamino acid racemase, beta-methylaspartate ammonia-lyase, and o-succinylbenzoate synthase. Comparative analysis of structure-function relationships within the superfamily suggests that carboxyphosphonoenolpyruvate synthase, another member of the superfamily, does not catalyze the reaction proposed in the literature but catalyzes an enolase-like reaction instead. The established and deduced structure-function relationships in the superfamily allow the prediction that other apparent members of the family for which no catalytic functions have yet been assigned will also perform chemistry involving abstraction of the alpha-protons of carboxylic acids.
Amino Acid Sequence↗
Exploration of the relationship between tetrachlorohydroquinone dehalogenase and the glutathione S-transferase superfamily.
Tetrachlorohydroquinone dehalogenase is found in Sphingomonas chlorophenolica, a soil bacterium that degrades pentachlorophenol, a widely used wood preservative. This enzyme converts tetrachlorohydroquinone (TCHQ) to trichlorohydroquinone (TriCHQ) and TriCHQ to dichlorohydroquinone (DCHQ) (Xun et al. (1992) J. Bacteriol. 174, 8003-8007). The reducing equivalents for each step are provided by two molecules of glutathione (Xun et al. (1992) Biochem. Biophys. Res. Commun. 182, 361-366). In addition to the expected TriCHQ and DCHQ products, the enzyme also produces substantial amounts of 2,3,5-trichloro-6-S-glutathionylhydroquinone (GS-TriCHQ) and an unidentified isomer of dichloro-S-glutathionylhydroquinone (GS-DCHQ). Treatment of the purified enzyme with dithiothreitol dramatically decreases the formation of GS-TriCHQ and GS-DCHQ. Furthermore, enzyme in freshly-prepared crude extracts forms only very small amounts of GS-TriCHQ and GS-DCHQ. We conclude that GS-TriCHQ and GS-DCHQ are produced by enzyme that has undergone some type of oxidative damage and are therefore not physiologically relevant products. The fact that the oxidative damage can be repaired by DTT suggests that a cysteine or methionine residue may be involved. We have created the C13S and C156S mutants of the enzyme. The C13S mutant converts TCHQ to GS-TriCHQ and GS-DCHQ, rather than to DCHQ. Thus, Cys13 is required for the reductive dehalogenation of TCHQ. A mechanism for the reaction which involves Cys13 is proposed.
Amino Acid Sequence↗
Sequence verification of human creatine kinase (43 kDa) isozymes by high-resolution tandem mass spectrometry.
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Amino Acid Sequence↗
Sequence verification of human creatine kinase (43 kDa) isozymes by high-resolution tandem mass spectrometry.
Amino acid sequencing by recombinant DNA technology, although dramatically useful, is subject to base reading errors, is indirect, and is insensitive to posttranslational processing. Mass spectrometry techniques can provide molecular weight data from even relatively large proteins for such cDNA sequences and can serve as a check of an enzyme's purity and sequence integrity. Multiply-charged ions from electrospray ionization can be dissociated to yield structural information by tandem mass spectrometry, providing a second method for gaining additional confidence in primary sequence confirmation. Here, accurate (+/- 1 Da) molecular weight and molecular ion dissociation information for human muscle and brain creatine kinases has been obtained by electrospray ionization coupled with Fourier-transform mass spectrometry to help distinguish which of several published amino acid sequences for both enzymes are correct. The results herein are consistent with one published sequence for each isozyme, and the heterogeneity indicated by isoelectric focusing due to 1-Da deamidation changes. This approach appears generally useful for detailed sequence verification of recombinant proteins.
Artifacts↗
A novel activity of OmpT. Proteolysis under extreme denaturing conditions.
A novel property of the bacterial outer membrane protein T, OmpT, has been discovered. It is active under extreme denaturing conditions. This finding emerged during characterization of a protease associated with the degradation of recombinant proteins expressed as inclusion bodies in Escherichia coli. These inclusion body proteins are stable to proteolytic degradation until they are solubilized by denaturation. The protease that degrades them under denaturing conditions was identified as OmpT on the basis of substrate specificity, inhibitor profile, and confirmation that its N-terminal sequence is identical with that of OmpT. A previously unknown property of this enzyme, OmpT's preference for denatured substrates, may provide a clue to its physiological function. To facilitate further characterization of this proteolytic activity, we have optimized a system to extract and assay OmpT under denaturing conditions using a soluble substrate, rabbit muscle creatine kinase.
Amino Acid Sequence↗
A functionally diverse enzyme superfamily that abstracts the alpha protons of carboxylic acids.
Mandelate racemase and muconate lactonizing enzyme are structurally homologous but catalyze different reactions, each initiated by proton abstraction from carbon. The structural similarity to mandelate racemase of a previously unidentified gene product was used to deduce its function as a galactonate dehydratase. In this enzyme superfamily that has evolved to catalyze proton abstraction from carbon, three variations of homologous active site architectures are now represented: lysine and histidine bases in the active site of mandelate racemase, only a lysine base in the active site of muconate lactonizing enzyme, and only a histidine base in the active site of galactonate dehydratase. This discovery supports the hypothesis that new enzymatic activities evolve by recruitment of a protein catalyzing the same type of chemical reaction.
Amino Acid Sequence↗
On the origins and functions of the enzymes of the 4-chlorobenzoate to 4-hydroxybenzoate converting pathway.
This review examines the enzymes of 4-chlorobenzoate to 4-hydroxybenzoate converting pathway found in certain soil bacteria. This pathway consists of three enzymes: 4-chlorobenzoate: Coenzyme A ligase, 4-chlorobenzoyl-Coenzyme A dehalogenase and 4-hydroxybenzoyl-Coenzyme A thioesterase. Recent progress made in the cloning and expression of the pathway genes from assorted bacterial strains is described. Gene order and sequence found among these strains are compared to reveal independent enzyme recruitment strategies. Sequence alignments made between the Pseudomonas sp. strain CBS3 4-chlorobenzoate pathway enzymes and structurally related proteins contained within the protein sequence data banks suggest possible origins in preexisting beta-oxidation pathways. The purification and characterization of the physical and kinetic properties of the pathway enzymes are described. Where possible a comparison of these properties between like enzymes from different bacterial sources are made.
Amino Acid Sequence↗
A novel structural basis for membrane association of a protein: construction of a chimeric soluble mutant of (S)-mandelate dehydrogenase from Pseudomonas putida.
The (S)-mandelate dehydrogenase (MDH) from Pseudomonas putida (ATCC 12633) is the only membrane-associated member of a homologous family of FMN-dependent, alpha-hydroxy acid dehydrogenases/oxidases that includes the structurally characterized glycolate oxidase from spinach (GOX). We have correlated the membrane association of MDH to a polypeptide segment in the interior of the primary sequence. This has been accomplished by construction of a chimeric enzyme in which the putative membrane-binding segment in MDH has been deleted and replaced with the corresponding segment from the soluble GOX. The resulting chimera, MDH-GOX, is soluble and retains partial catalytic activity (approximately 1%) using (S)-mandelate as substrate. In contrast, the activities of both the membrane-associated wild-type MDH and the soluble MDH-GOX are nearly the same when (S)-phenyllactate is used as substrate. To the best of our knowledge, this is the first example of a membrane-associated protein in which an internal polypeptide segment anchors the protein to the membrane.
Alcohol Oxidoreductases↗
3-Carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida is homologous to the class II fumarase family: a new reaction in the evolution of a mechanistic motif.
The gene (pcaB) for 3-carboxymuconate lactonizing enzyme (CMLE; 3-carboxymuconate cycloisomerase; EC 5.5.1.2) from Pseudomonas putida has been cloned into pMG27NS, a temperature-sensitive expression vector, and expressed in Escherichia coli N4830. The specific activity and kinetic parameters of the recombinant CMLE were comparable to those previously reported. A comparison of the deduced amino acid sequence of CMLE with sequences available in the PIR and Genbank databases revealed that CMLE has highly significant sequence homology to the class II fumarase family, particularly to adenylosuccinate lyase from Bacillus subtilis. CMLE has no significant homology to muconate lactonizing enzyme (MLE) from P. putida, its sister enzyme in the beta-ketoadipate pathway. These findings fully corroborate a prediction made by us on the basis of mechanistic and stereochemical analyses of CMLE and MLE [Chari, R. V. J., Whitman, C. P., Kozarich, J. W., Ngai, K.-L., & Ornston, L. N. (1987) J. Am. Chem. Soc. 109, 5514-5519] and suggest that CMLE and MLE were recruited into this specialized pathway from two different enzyme families.
Amino Acid Sequence↗
Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases.
We have deduced the nucleotide sequence of the genes encoding the three components of 4-chlorobenzoate (4-CBA) dehalogenase from Pseudomonas sp. CBS-3 and examined the origin of these proteins by homology analysis. Open reading frame 1 (ORF1) encodes a 30-kDa 4-CBA-coenzyme A dehalogenase related to enoyl-coenzyme A hydratases functioning in fatty acid beta-oxidation. ORF2 encodes a 57-kDa protein which activates 4-CBA by acyl adenylation/thioesterification. This 4-CBA:coenzyme A ligase shares significant sequence similarity with a large group of proteins, many of which catalyze similar chemistry in beta-oxidation pathways or in siderophore and antibiotic synthetic pathways. These proteins have in common a short stretch of sequence, (T,S)(S,G)G(T,S)(T,E)G(L,X)PK(G,-), which is particularly highly conserved and which may represent an important new class of "signature" sequence. We were unable to find any proteins homologous in sequence to the 16-kDa 4-hydroxybenzoate-coenzyme A thioesterase encoded by ORF3. Analysis of the chemistry and function of the proteins found to be structurally related to the 4-CBA:coenzyme A ligase and the 4-CBA-coenzyme A dehalogenase supports the proposal that they evolved from a beta-oxidation pathway.
Amino Acid Sequence↗
The principal islet of the Coho salmon (Oncorhyncus kisutch) contains the BB isoenzyme of creatine kinase.
The importance of creatine kinase (E.C. 2.7.3.2) in endocrine tissues has been generally overlooked. Using a specific radiometric assay, we have demonstrated the existence of CK in the Brockmann body (principal islet) of the Coho salmon. We have purified this protein from insular tissue and concurrently purified CK from brain and muscle of the salmon. Purification characteristics, immunological cross-reactivity, and N-terminal sequence analysis have demonstrated that the predominant cytosolic CK from the Brockmann body is indistinguishable from the BB (brain) isoenzyme. Immunocytochemical studies indicated that the enzyme resides in the endocrine parenchyma. Phosphocreatine may serve as a reservoir of energy in the islet and augment its capacity to secrete hormones. The induction of CK-BB in the islet by other hormones could influence the secretion of insular hormones. Interorgan flux of the substrate creatine may be an undescribed mechanism of physiological regulation.
Amino Acid Sequence↗
Novel enzymic hydrolytic dehalogenation of a chlorinated aromatic.
Microbial enzyme systems may be used in the biodegradation of persistent environmental pollutants. The three polypeptide components of one such system, the 4-chlorobenzoate dehalogenase system, have been isolated, and the chemical steps of the 4-hydroxybenzoate-forming reaction that they catalyze have been identified. The genes contained within a 4.5-kilobase Pseudomonas sp. strain CBS3 chromosomal DNA fragment that encode dehalogenase activity were selectively expressed in transformed Escherichia coli. Oligonucleotide sequencing revealed a stretch of homology between the 57-kilodalton (kD) polypeptide and several magnesium adenosine triphosphate (MgATP)-cleaving enzymes that allowed MgATP and coenzyme A (CoA) to be identified as the dehalogenase cosubstrate and cofactor, respectively. The dehalogenase activity arises from two components, a 4-chlorobenzoate:CoA ligase-dehalogenase (an alpha beta dimer of the 57- and 30-kD polypeptides) and a thioesterase (the 16-kD polypeptide).
Adenosine Triphosphate↗
Cloning and expression of functional rabbit muscle creatine kinase in Escherichia coli. Addressing the problem of microheterogeneity.
The gene encoding rabbit muscle creatine kinase (CK) has been subcloned into a single plasmid, and the protein expressed in a soluble and functional form in Escherichia coli. The amino terminus, specific activity, and electrophoretic mobility of the E. coli-expressed creatine kinase are all identical with that of creatine kinase purified from rabbit skeletal muscle. Surprisingly, isoelectric focusing shows that the expressed protein displays no less heterogeneity than the tissue-purified material. The identification of the source(s) of this heterogeneity is important for the preparation of highly homogeneous material needed for structural studies and clinical applications. This issue also has implications for studies of the developmental regulation and tissue localization of the various CK genes. Our results allow us to eliminate some of the proposals, such as the presence of multiple alleles, alternative ribosomal initiation sites, and post-translational glycosylation or phosphorylation that have been suggested to explain the presence of the numerous isoforms present in apparently pure preparations of CK.
Animals↗
Analysis of sequence homologies in plant and bacterial pyruvate phosphate dikinase, enzyme I of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and other PEP-utilizing enzymes. Identification of potential catalytic and regulatory motifs.
In this paper we report the amino acid sequence of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus as determined from the nucleotide sequence of the PPDK gene. Comparison of the B. symbiosus PPDK amino acid sequence with that of the maize PPDK [Matsuoka, M., Ozeki, Y., Yamamoto, N., Hirano, H., Kamo-Murakami, Y., & Tanaka, Y. (1988) J. Biol. Chem. 263, 11080] revealed long stretches of homologous sequence (greater than 70% identity), which contributed to an overall sequence identity of 53%. The circular dichrosim spectra, hydropathy profiles, and calculated secondary structural elements of the two dikinases suggest that they may have very similar tertiary structures as well. A comparison made between the amino acid sequence of the maize and B. symbiosus dikinase with other known protein sequences revealed homology, concentrated in three stretches of sequences, to a mechanistically related enzyme, enzyme I of the Escherichia coli PEP: sugar phosphotransferase system [Saffen, D. W., Presper, K. A., Doering, T. L., Roseman, S. (1987) J. Biol. Chem. 262, 16241]. It is proposed that (i) these three stretches of sequence constitute the site for PEP binding and catalysis and a possible site for the regulation of enzymatic activity and (ii) the conserved sequences exist in a third mechanistically related enzyme, PEP synthase.
Amino Acid Sequence↗
Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli.
The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.
Acetolactate Synthase↗
Removal of a proteolytic activity associated with aggregates formed from expression of creatine kinase in Escherichia coli leads to improved recovery of active enzyme.
Expression of creatine kinase (CK) from a Torpedo californica electric organ cDNA in Escherichia coli results in an insoluble protein product with no detectable CK activity. Although this is a stable aggregate that can be isolated in an enriched form by centrifugation, initial attempts to generate enzyme activity by denaturing and refolding yielded only minute amounts of active protein. We find that these low recoveries are due to proteolysis of the CK during denaturation and refolding. While this proteolytic activity is not inhibited by either phenylmethanesulfonyl fluoride (PMSF) or EDTA, it can be largely removed from the CK aggregate by extraction with a detergent-containing buffer prior to denaturation. This treatment improves the recovery of active CK approximately 100-fold. We have also found similar proteolytic activity associated with the aggregate formed when a mutant of bovine pancreatic trypsin inhibitor (BPTI) is expressed in E. coli. Discovery of this proteolytic activity in two different expression systems suggests that it should be considered as a potential problem for recovery of active protein from other inclusion bodies as well.
Animals↗