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PubMed2002

Organization of the multifunctional enzyme type 1: interaction between N- and C-terminal domains is required for the hydratase-1/isomerase activity.

Rat peroxisomal multifunctional enzyme type 1 (perMFE-1) is a monomeric protein of beta-oxidation. We have defined five functional domains (A, B, C, D and E) in the perMFE-1 based on comparison of the amino acid sequence with homologous proteins from databases and structural data of the hydratase-1/isomerases (H1/I) and (3 S )-hydroxyacyl-CoA dehydrogenases (HAD). Domain A (residues 1-190) comprises the H1/I fold and catalyses both 2-enoyl-CoA hydratase-1 and Delta(3)-Delta(2)-enoyl-CoA isomerase reactions. Domain B (residues 191-280) links domain A to the (3 S )-dehydrogenase region, which includes both domain C (residues 281-474) and domain D (residues 480-583). Domains C and D carry features of the dinucleotide-binding and the dimerization domains of monofunctional HADs respectively. Domain E (residues 584-722) has sequence similarity to domain D of the perMFE-1, which suggests that it has evolved via partial gene duplication. Experiments with engineered perMFE-1 variants demonstrate that the H1/I competence of domain A requires stabilizing interactions with domains D and E. The variant His-perMFE (residues 288-479)Delta, in which the domain C is deleted, is stable and has hydratase-1 activity. It is proposed that the extreme C-terminal domain E in perMFE-1 serves the following three functions: (i) participation in the folding of the N-terminus into a functionally competent H1/I fold, (ii) stabilization of the dehydrogenation domains by interaction with the domain D and (iii) the targeting of the perMFE-1 to peroxisomes via its C-terminal tripeptide.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1997

Peroxisomal multifunctional enzyme of beta-oxidation metabolizing D-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization.

In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase-hydratase-dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of beta-oxidation but through reciprocal stereochemical courses.

Amino Acid Sequence↗

PubMed1995

Arrangement of catalytic sites in the multifunctional enzyme enniatin synthetase.

Enniatin synthetase is an N-methyl peptide synthetase comprising 3131 amino acids. Catalytic sites of the 347-kDa multifunctional enzyme were mapped by N-terminal sequencing of substrate affinity-labelled enzyme fragments formed by proteolysis, and functional studies of purified enniatin synthetase fragments. An N-terminal 200-kDa fragment containing the cofactor 4'-phosphopantetheine was able to activate D-hydroxyisovaleric acid (D-HOiVl) as a thioester. The N-termini of two [14C]HOiVl-labelled enzyme fragments could be assigned to amino acid position 429 within the N-terminal conserved enniatin synthetase portion named EA. This portion of about 600 amino acids shares high similarity to microbial peptide synthetase regions. A 68-kDa L-[14C]Val-labelled enniatin synthetase fragment was localized at amino acid position 2294 within the second C-terminal conserved protein portion EB. Additionally enniatin synthetase was labelled with isovaleryl-L-[14C]Val, an analogue of the D-hydroxyisovaleryl-L-Val intermediate in enniatin biosynthesis. The N-terminus of a 30-kDa isovaleryl-L-[14C]Val-labelled enniatin synthetase fragment was mapped in a C-terminal segment of the protein portion EA. The same N-terminal sequence was obtained from a 60-kDa enniatin synthetase fragment modified with [3H]beta Ala, a constituent of the cofactor 4'-phosphopantetheine. This indicates the presence of the cofactor in this protein fragment. Localization of the methyltransferase function of enniatin synthetase in an amino acid portion integrated into region EB was achieved by N-terminal sequencing of a photolabelled S-[methyl-14C]adenosyl methionine 45-kDa fragment and the identification of a photolabelled peptide Asn-Leu-Asn-Pro-Gly-Leu-Asn-Ser-Tyr.

Amino Acid Sequence↗

PubMed2003

Crystallization and preliminary crystallographic data of 2-enoyl-CoA hydratase 2 domain of Candida tropicalis peroxisomal multifunctional enzyme type 2.

In yeast, the second and the third reaction of the fatty-acid beta-oxidation spiral are catalysed by peroxisomal multifunctional enzyme type 2 (Mfe2p/Fox2p). This protein has two (3R)-hydroxyacyl-CoA dehydrogenase domains and a C-terminal 2-enoyl-CoA hydratase 2 domain. Here, the purification, crystallization and X-ray diffraction analysis of the hydratase 2 domain [CtMfe2p(dh(a+b)Delta)] from Candida tropicalis Mfe2p is reported. CtMfe2p(dh(a+b)Delta) was overexpressed as an enzymatically active recombinant protein and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to space group C2, with unit-cell parameters a = 178.57, b = 60.46, c = 130.85 A, beta = 94.48 degrees. Selenomethionine-labelled protein was used for a multi-wavelength anomalous dispersion (MAD) experiment. A three-wavelength data set suitable for MAD phasing was collected to 2.25 A resolution using synchrotron radiation.

Candida tropicalis↗

PubMed2001

Crystal structure of the liganded SCP-2-like domain of human peroxisomal multifunctional enzyme type 2 at 1.75 A resolution.

beta-Oxidation of amino acyl coenzyme A (acyl-CoA) species in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. MFE-2 has a modular organization of three domains. The function of the C-terminal domain of the mammalian MFE-2, which shows similarity with sterol carrier protein type 2 (SCP-2), is unclear. Here, the structure of the SCP-2-like domain comprising amino acid residues 618-736 of human MFE-2 (d Delta h Delta SCP-2L) was solved at 1.75 A resolution in complex with Triton X-100, an analog of a lipid molecule. This is the first reported structure of an MFE-2 domain. The d Delta h Delta SCP-2L has an alpha/beta-fold consisting of five beta-strands and five alpha-helices; the overall architecture resembles the rabbit and human SCP-2 structures. However, the structure of d Delta h Delta SCP-2L shows a hydrophobic tunnel that traverses the protein, which is occupied by an ordered Triton X-100 molecule. The tunnel is large enough to accommodate molecules such as straight-chain and branched-chain fatty acyl-CoAs and bile acid intermediates. Large empty apolar cavities are observed near the exit of the tunnel and between the helices C and D. In addition, the C-terminal peroxisomal targeting signal is ordered in the structure and solvent-exposed, which is not the case with unliganded rabbit SCP-2, supporting the hypothesis of a ligand-assisted targeting mechanism.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1999

Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid.

The yeast peroxisomal (3R)-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase 2 (multifunctional enzyme type 2; MFE-2) has two N-terminal domains belonging to the short chain alcohol dehydrogenase/reductase superfamily. To investigate the physiological roles of these domains, here called A and B, Saccharomyces cerevisiae fox-2 cells (devoid of Sc MFE-2) were taken as a model system. Gly(16) and Gly(329) of the S. cerevisiae A and B domains, corresponding to Gly(16), which is mutated in the human MFE-2 deficiency, were mutated to serine and cloned into the yeast expression plasmid pYE352. In oleic acid medium, fox-2 cells transformed with pYE352:: ScMFE-2(aDelta) and pYE352::ScMFE-2(bDelta) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(aDeltabDelta) failed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE- 2(h2Delta)) and mutational variants of the A and B domains (Ct MFE- 2(h2DeltaaDelta), Ct MFE- 2(h2DeltabDelta), and Ct MFE- 2(h2DeltaaDeltabDelta)) were overexpressed and characterized. All proteins were dimers with similar secondary structure elements. Both wild type domains were enzymatically active, with the B domain showing the highest activity with short chain and the A domain with medium and long chain (3R)-hydroxyacyl-CoA substrates. The data show that the dehydrogenase domains of yeast MFE-2 have different substrate specificities required to allow the yeast to propagate optimally on fatty acids as the carbon source.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1992

Molecular cloning and sequencing of the gene for mycocerosic acid synthase, a novel fatty acid elongating multifunctional enzyme, from Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guerin.

Mycocerosyl lipids are found uniquely in the cell walls of pathogenic mycobacteria. Mycocerosic acid synthase (MAS) is a multifunctional protein which catalyzes elongation of n-fatty acyl-CoA with methylamalonyl-CoA as the elongating agent (Rainwater, D. L., and Kolattukudy, P. E. (1985) J. Biol. Chem. 260, 616-623). To understand how the various domains that catalyze the reactions involved in chain elongation are organized, mas gene from Mycobacterium tuberculosis bovis BCG was cloned. A lambda gt11 library of AluI partially digested genomic DNA from the organism was screened with an oligonucleotide probe designed from the N-terminal amino acid sequence of purified MAS. Using terminal segments of inserts from positive clones as the probe, the library was rescreened and the process was repeated. Sequencing of four overlapping clones revealed a contiguous sequence of 9699 base pair(s) (bp) of mycobacterial genome containing a 6330-bp open reading frame that could code for a protein of 2100 amino acids with a molecular mass of 225,437 daltons. The authenticity of the open reading frame as that of MAS was verified by correspondence of the amino acid sequences deduced from the gene with the directly determined amino acid sequences of the N terminus and three different internal peptide fragments. By comparing the MAS amino acid sequence with the sequences in the active site regions of known fatty acid synthases and polyketide synthases the functional domains in MAS were identified. This analysis showed that the domains were organized in the following order: beta-ketoacyl synthase, acyl transferase, dehydratase-enoyl reductase, beta-ketoreductase, acyl carrier protein; no thioesterase-like domain could be found. These results establish MAS as the first case of an elongating multifunctional enzyme composed of two identical subunits that resemble the vertebrate fatty acid synthase in size, subunit structure, and linear organization of functional domains. Southern and Western blot analyses showed absence of mas gene and encoded proteins in Mycobacterium smegmatis and Escherichia coli. This result is consistent with the report that mycocerosic acid is present only in pathogenic mycobacteria.

Acyltransferases↗

PubMed2000

Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity.

Beta-oxidation of acyl-CoAs in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. Amino acid sequence alignment of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s reveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-408, Tyr-410, Asp-490, Tyr-505, Asp-510, His-515, Asp-517, and His-532. To investigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were tested for complementation in a yeast. After additional screening, the wild type and noncomplementing E366A and D510A variants were expressed and characterized. The purified proteins have similar secondary structural elements, with the same subunit composition. The E366A variant had a k(cat)/K(m) value 100 times lower than that of the wild type MFE-2 at pH 5, whereas the D510A variant was inactive. Asp-510 was imbedded in a novel hydratase 2 motif found in the hydratase 2 proteins. The data show that the hydratase 2 reaction catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, suggesting that their catalytic role may be equivalent to that of the two catalytic residues of hydratase 1.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1999

Human brain short chain L-3-hydroxyacyl coenzyme A dehydrogenase is a single-domain multifunctional enzyme. Characterization of a novel 17beta-hydroxysteroid dehydrogenase.

Human brain short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) was found to catalyze the oxidation of 17beta-estradiol and dihydroandrosterone as well as alcohols. Mitochondria have been demonstrated to be the proper location of this NAD+-dependent dehydrogenase in cells, although its primary structure is identical to an amyloid beta-peptide binding protein reportedly associated with the endoplasmic reticulum (ERAB). This fatty acid beta-oxidation enzyme was identified as a novel 17beta-hydroxysteroid dehydrogenase responsible for the inactivation of sex steroid hormones. The catalytic rate constant of the purified enzyme was estimated to be 0.66 min-1 with apparent Km values of 43 and 50 microM for 17beta-estradiol and NAD+, respectively. The catalytic efficiency of this enzyme for the oxidation of 17beta-estradiol was comparable with that of peroxisomal 17beta-hydroxysteroid dehydrogenase type 4. As a result, the human SCHAD gene product, a single-domain multifunctional enzyme, appears to function in two different pathways of lipid metabolism. Because the catalytic functions of human brain short chain L-3-hydroxyacyl-CoA dehydrogenase could weaken the protective effects of estrogen and generate aldehydes in neurons, it is proposed that a high concentration of this enzyme in brain is a potential risk factor for Alzheimer's disease.

17-Hydroxysteroid Dehydrogenases↗

PubMed2000

Swinging arms and swinging domains in multifunctional enzymes: catalytic machines for multistep reactions.

Multistep chemical reactions are increasingly seen as important in a growing number of complex biotransformations. Covalently attached prosthetic groups or swinging arms, and their associated protein domains, are essential to the mechanisms of active-site coupling and substrate channeling in a number of the multifunctional enzyme systems responsible. The protein domains, for which the posttranslational machinery in the cell is highly specific, are crucially important, contributing to the processes of molecular recognition that define and protect the substrates and the catalytic intermediates. The domains have novel folds and move by virtue of conformationally flexible linker regions that tether them to other components of their respective multienzyme complexes. Structural and mechanistic imperatives are becoming apparent as the assembly pathways and the coupling of multistep reactions catalyzed by these dauntingly complex molecular machines are unraveled.

Animals↗

PubMed2003

Modification of the monomer composition of polyhydroxyalkanoate synthesized in Saccharomyces cerevisiae expressing variants of the beta-oxidation-associated multifunctional enzyme.

Expression by Saccharomyces cerevisiae of a polyhydroxyalkanoate (PHA) synthase modified at the carboxy end by the addition of a peroxisome targeting signal derived from the last 34 amino acids of the Brassica napus isocitrate lyase (ICL) and containing the terminal tripeptide Ser-Arg-Met resulted in the synthesis of PHA. The ability of the terminal peptide Ser-Arg-Met and of the 34-amino-acid peptide from the B. napus ICL to target foreign proteins to the peroxisome of S. cerevisiae was demonstrated with green fluorescent protein fusions. PHA synthesis was found to be dependent on the presence of both the enzymes generating the beta-oxidation intermediate 3-hydroxyacyl-coenzyme A (3-hydroxyacyl-[CoA]) and the peroxin-encoding PEX5 gene, demonstrating the requirement for a functional peroxisome and a beta-oxidation cycle for PHA synthesis. Using a variant of the S. cerevisiae beta-oxidation multifunctional enzyme with a mutation inactivating the B domain of the R-3-hydroxyacyl-CoA dehydrogenase, it was possible to modify the PHA monomer composition through an increase in the proportion of the short-chain monomers of five and six carbons.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1999

Multifunctional enzymes and evolution of biosynthetic pathways: retro-evolution by jumps.

A likely scenario of evolution of biosynthetic pathways is believed to have occurred by retro-evolution through recruitment of existing enzymes rather than generation of de novo classes. It had been proposed that such retro-evolution occurred in steps as a response to depletion of an essential metabolite and availability of another related substance in the environment. In this article, I argue that because of instability of many such extant intermediates, it is unlikely that retro-evolution had occurred in steps. I further propose that such evolution in many cases has taken place by jumps, i.e., by recruitment of a multifunctional enzyme capable of catalyzing several steps at a time, albeit inefficiently. I further speculate that in some cases one primordial multienzyme may have catalyzed the whole sequence of reaction of a biosynthetic pathway, i.e., the pathway may have evolved by a single leap. Gene duplications and further evolution to more efficient enzymes led to extant pathways. Such a mechanism predicts that some or all enzymes of a pathway must have descended from a common ancestor. Sequence and structural homologies among extant enzymes of a biosynthetic pathway have been examined.

Amino Acids↗

PubMed2003

Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of alpha-3',4'-anhydrovinblastine: a specific role for a multifunctional enzyme.

We have characterized a basic peroxidase with alpha-3',4'-anhydrovinblastine (AVLB) synthase activity, which was purified from Catharanthus roseus leaves. This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves, and the single AVLB synthase activity detected in C. roseus extracts. It was observed that the monomeric substrates of AVLB, vindoline and catharanthine, are both suitable electron donors for the oxidizing intermediates of the basic peroxidase, compounds I and II. Results also showed that the reaction proceeds by a radical-propagated mechanism. Substrate specificity studies of the enzyme revealed that it was also able to oxidize several common peroxidase substrates, indicating a broad range of substrate specificity that is characteristic of class III plant peroxidases. Cytochemical studies showed that the enzyme is localized in C. roseus mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. This particular location suggests a meaningful spatial organization that led to the proposal of a metabolic channeling model for the peroxidase-mediated synthesis of AVLB. The importance of this type of mechanism in the regulation of peroxidase isoenzyme functions in vivo is discussed. In view of the results obtained it is concluded that the basic peroxidase present in C. roseus leaves fulfills all the requirements to be considered as an AVLB synthase, and it is proposed that this specific function of this multifunctional enzyme is determined by metabolic channeling resulting from specific protein-protein interactions.

Catharanthus↗

PubMed1983

Interaction of tetrahydropteroylpolyglutamates with two folate-dependent multifunctional enzymes.

The naturally occurring pteroylpolyglutamate derivatives are substrates for the folate-mediated reactions in cells, including the reactions catalyzed by two multifunctional folate dependent enzymes in eucaryotes. The appropriate derivatives of tetrahydropteroyl (glutamate)n where n = 1, 3, 5, or 7 were used to determine the specificity for, and kinetic advantages of the extra glutamyl residues with two multifunctional proteins from pig liver: methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase, and formiminotransferase-formininotetrahydrofolate cyclodeaminase. Specificity for the polyglutamate derivatives ranged from 10- to 70-fold as indicated from Km values or from the ability to inhibit the five different enzyme activities. With the sequential activities of the transferase-deaminase enzyme, it was demonstrated that when the tetrahydropteroyl pentaglutamate is used as a substrate, the intermediate formimino-compound does not accumulate in the medium. That this kinetic observation is due to preferential transfer of the pentaglutamate- but not monoglutamate intermediate from transferase to deaminase sites without its release from the enzyme molecule was supported by three types of experiments. Chemical modification to yield monofunctional derivatives of the transferase-deaminase affected the kinetics of the recombined activities only with the pentaglutamate substrate, causing a lag in the appearance of final product. Inhibition studies demonstrated that the deaminase activity could preferentially be inhibited only with the monoglutamate substrate. The deaminase activity with the monoglutamate substrate was increased by providing elevated formiminotetrahydrofolate in the assay mixture; no effect was observed when the reaction was carried out with pentaglutamate. Preliminary binding studies indicate a single folate site per subunit of the octameric enzyme, suggesting a type of combined transferase-deaminase site.

Aminohydrolases↗

PubMed1976

Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase, from human erythrocytes. Evidence for a common active site.

Bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities responsible for 2,3-bisphosphoglycerate metabolsim in human red cells are displayed by the same enzyme protein which has phosphoglyceromutase activity [Sasaki, R., et al. (1975) Eur J. Biochem. 50, 581-593]. This enzyme was subjected to chemical modification by trinitrobenzenesulfonate. The three enzyme activities were inactivated by trinitrobenzenesulfonate at the same rate. The sulfhydryl content of the enzyme was unchanged during trinitrophenylation, indicating that derivatization was through the amino group. Trinitrophenylation of about one amino group per mole of the enzyme resulted in complete loss of the three activities. Both 2,3-bisphosphoglycerate and 1,3-bisphosphoglycerate inhibited trinitrophenylation and effectively protected the enzyme from inactivation. Although monophosphoglycerates did not show any protective effect at concentrations which should be adequate based upon their kinetic constants, they were protective at higher concentrations. Inactivation by trinitrophenylation was an apparent first-order reaction. The dissociation constant of the enzyme - 2,3-bisphosphoglycerate complex was determined by analyzing the first-order reaction on the assumption that the protective effect of 2,3-bisphosphoglycerate was due to competition with trinitrobenzenesulfonate. The dissociation constant was in good agreement with kinetic constants of 2,3-bisphosphoglycerate in the enzyme reactions, which indicated that 2,3-bisphosphoglycerate did indeed exert its protective effect through competition with trinitrobenzenesulfonate for an amino group of the enzyme. The protective effect of monophosphoglycerates could be rationalized with kinetic evidence that 2-phosphoglycerate at high concentrations interacts with the 2,3-bisphosphoglycerate binding site. These results indicate that the enzyme exhibits the three enzyme activities at a common active site at which one amino group essential for binding of bisphosphoglycerates is located. Based on the multifunctional properties of this enzyme, a possible mechanism was discussed for regulation of 2,3-bisphosphoglycerate metabolism in human red cells.

Binding Sites↗

PubMed1989

Molecular characterization of a Dictyostelium discoideum gene encoding a multifunctional enzyme of the pyrimidine pathway.

We have isolated and characterized a Dictyostelium discoideum gene (PYR1-3) encoding a multifunctional protein that carries the three first enzymatic activities of the de novo pyrimidine biosynthetic pathway. The PYR1-3 gene is adjacent to another gene of the pyrimidine biosynthetic pathway (PYR4); the two genes are separated by a 1.5-kb non-coding sequence and transcribed divergently. The PYR1-3 gene is transcribed to form a 7.5-kb polyadenylated mRNA. As with the other genes of the pyrimidine biosynthetic pathway, the PYR1-3 mRNA level is high during growth and decreases sharply during development. We have determined the nucleotide sequence of 63% of the coding region of the PYR1-3 gene. We have identified the activities of the protein encoded by the D. discoideum PYR1-3 gene by comparison of amino acid sequences with the products of genes of known function. The PYR1-3 gene contains four distinct regions that probably correspond to four domains in the protein. From the NH2 extremity to the COOH extremity, these domains are: glutamine amidotransferase, carbamoylphosphate synthetase, dihydroorotase and aspartate transcarbamylase. This organization is identical to the one found in the rudimentary gene of Drosophila. The evolutionary implications of this finding are discussed.

Amino Acid Sequence↗

PubMed2002

Arbutin synthase, a novel member of the NRD1beta glycosyltransferase family, is a unique multifunctional enzyme converting various natural products and xenobiotics.

Plant glucosyltransferases (GTs) play a crucial role in natural product biosynthesis and metabolization of xenobiotics. We expressed the arbutin synthase (AS) cDNA from Rauvolfia serpentina cell suspension cultures in Escherichia coli with a 6 x His tag and purified the active enzyme to homogeneity. The recombinant enzyme had a temperature optimum of 50 degrees C and showed two different pH optima (4.5 and 6.8 or 7.5, depending on the buffer). Out of 74 natural and synthetic phenols and two cinnamyl alcohols tested as substrates for the AS, 45 were accepted, covering a broad range of structural features. Converting rates comparable to hydroquinone were not achieved. In contrast to this broad acceptor substrate specificity, only pyrimidine nucleotide activated glucose was tolerated as a donor substrate. Nucleotide and amino acid sequence analysis revealed AS to be a new member of the NRD1beta family of glycosyl transferases and placed the enzyme into the group of plant secondary product GTs. Arbutin synthase is therefore the first example of a broad spectrum multifunctional glucosyltransferase.

Amino Acid Sequence↗

PubMed1992

Enzymatic characterisation of the multifunctional enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, the multienzyme catalyzing the formation of ACV from the constituent amino acids and ATP in the presence of Mg2+ and dithioerythritol, was purified about 2700-fold from Streptomyces clavuligerus. The molecular mass of the native enzyme as determined by gel filtration chromatography is 560 kDa, while that determined by denaturing gel electrophoresis is 500 kDa. The enzyme is able to catalyze pyrophosphate exchange in dependence on L-cysteine and L-valine, but no L-alpha-aminoadipic-acid-dependent ATP/PPi exchange could be detected. Other L-cysteine- and L-valine-activating enzymes present in crude extracts were identified as aminoacyl-tRNA synthetases which could be separated from ACV synthetase. The molecular mass of these enzymes is 140 kDa for L-valine ligase and 50 kDa for L-cysteine ligase. The dissociation constants have been estimated, assuming three independent activation sites, to be 1.25 mM and 1.5 mM for cysteine and ATP, and 2.4 mM and 0.25 mM for valine and ATP, respectively. The enzyme forms a thioester with alpha-aminoadipic acid and with valine in a molar ratio of 0.6:1 (amino acid/enzyme). Thus, the bacterial ACV synthetase is a multifunctional peptide synthetase, differing from fungal ACV synthetases in its mechanism of activation of the non-protein amino acid.

Amino Acyl-tRNA Synthetases↗