Isoforms of dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24: role of the signal sequence and overexpression in Escherichia coli

The complete nucleotide sequence of dipeptidyl aminopeptidase IV (DAP IV) from Pseudomonas sp. WO24 was determined. Nucleotide sequence analysis revealed an open reading frame of 2238bp, which was assigned to dap4 by N-terminal and internal amino acid sequences previously reported. The predicted amino acid sequence of DAP IV contains a serine protease Gly-X-Ser-X-Gly-Gly consensus motif and displays extensive homology to DAP IVs and the homologous proteins from eukaryotes and bacteria, belonging to the prolyl oligopeptidase family S9. In Pseudomonas sp. WO24, DAP IV is expressed as 82 and 84-kDa isoforms, having two Met, Met-1 and Met-12, in its N-terminal sequence. Met-1 of DAP IV was mutated to Gly and Met-12 was mutated to Ile, and we overexpressed the two mutated genes in Escherichia coli and obtained the recombinant 82 and 84-kDa proteins from the periplasm and the cytoplasm, respectively, suggesting that the 82 and 84-kDa isoforms are derived from the same gene and localize to different compartments in the cell. We developed purification steps for activting a large amount of 84-kDa isoform protein that will be useful for producing protein for crystallographic studies.     

Peptidases in the kidney of male rats following castration and testosterone substitution

The localization of aminopeptidase M (APM), dipeptidyl peptidase I (DAP I), II (DAP II) and IV (DAP IV) in the renal section was investigated histochemically, and their activities were determined fluorometrically in renal homogenate of normal, castrated and testosteron treated male rats.--After castration the activities of the lysosomal DAP II (pars convoluta of the proximal tubule), DAP I (distal and proximal tubule) and of the mainly membrane-bound DAP IV (glomeruli, brush border of the proximal tubule) increase in comparison to normal males, whereas the activities of the brush border-bound APM decrease. After testosteron treatment of castrated animals (0.1, 0.5 and 1.0 mg testosterone proprionate/100 g BW and day; 5-day treatment) the activities of DAP I, II and IV decrease again, so that after treatment with 0.1 mg testosterone proprionate, the activities of DAP I and II approach those in normal males.--The additionally determined urinary protein excretion shows that there is a significant decrease in proteinuria after castration, whereas testosterone treatment of castrated animals is accompanied by an increase of proteinuria.--Our results would suggest that the protein catabolism in the proximal tubule and the proteinuria are interrelated, and that testosterone influences (decreases) the protein catabolism in the proximal tubule. This means that high activities of lysosomal proteinases in the proximal tubule (castrates) are accompanied by a low proteinuria, and low activities of those proteinases (testosterone treated castrated or normal males) by a high proteinuria.     

Reconstitution of mature plastocyanin from precursor apo-plastocyanin expressed in Escherichia coli

The precursor plastocyanin from Silene pratensis (white campion) has been expressed in Escherichia coli. The precursor protein was accumulated in insoluble aggregates and partially purified as an apo-protein. The purified precursor apo-plastocyanin was processed to the mature apo-plastocyanin by chloroplast extracts. N-terminal amino-acid sequencing indicated that the processed protein was identical to the N-terminal amino-acid residues of mature plastocyanin that was deduced from the nucleotide sequence. The copper could be incorporated into the apo-plastocyanin of mature size in vitro, but could not into the precursor apo-plastocyanin under the same conditions. Absorption spectra and reduction potential of the reconstituted mature plastocyanin were indistinguishable from those of the purified spinach plastocyanin. The electron transfer activities of the reconstituted plastocyanin with both the Photosystem I reaction center (P700) and cytochrome f were almost the same as those of the purified spinach plastocyanin.     

Hydrolysis of polypeptides and proteins utilizing a mixture of dipeptidylaminopeptidases with analysis by GC/MS.

The hydrolysis of polypeptides and proteins to dipeptides using a mixture of dipeptidylaminopeptidase I and IV is demonstrated for several compounds. The dipeptide products were identified by gas chromatography/mass spectrometry of their acyl, ester derivatives. Partial hydrolysis of carboxymethylated soybean trypsin inhibitor (mw 20,327) shows the viability of the methods with an intact protein. Polypeptides with N-terminal arginyl or lysyl residues, which cannot be hydrolyzed by dipeptidylaminopeptidase I alone, are readily hydrolyzed by the dipeptidylaminopeptidase $\text{I}\text{IV}$ mixture, as shown by the digestion of ribonuclease S-peptide. The enzyme mixture releases dipeptides from the N-terminus of a polypeptide or protein with hydrolysis being terminated only when proline appears in position three of a remaining polypeptide fragment. The techniques are discussed in terms of new types of analyses which can be performed using this combined enzyme-GC/MS method.     

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates.

It is suggested that besides the reaction sequence cytochrome 553 → plastocyanin → Photosystem I reaction center, a second pathway cytochrome 553 → Photosystem I reaction center may operate additionally.     

Studies on dipeptidyl(amino)peptidase IV (glycyl-proline naphthylamidase)

Summary The activity of dipeptidyl(amino)peptidase IV (DAP IV, glycylproline naphthylamidase) was discovered in the endothelial cells of the venous part of capillary bed and of small venules of many organs of the rat, mini-pig, rabbit, cock as well as man. In aortae, large arteries and veins only a portion of vase vasorum displays a positive reaction. Glycyl-proline-4-methoxy-2-naphthylamide (Gly-Pro-MNA) is the substrate of choice both from the viewpoint of enzyme kinetics as well as localization. Phenylalanyl-proline-4-methoxy-2-naphthylamide (Phe-Pro-MNA) is cleaved less easily, however, it enables a good localization. 1- and 2-naphthylamine derivatives of glycylproline display better kinetic properties than Phe-Pro-MNA, however they enable a satisfactory localization under special conditions only.The recommended diazonium salt for the routine is Fast Blue B. The enzyme is quite firmly associated with the structure and chloroform-acetone preextraction of cryostat sections does not influence its activity significantly while improving the localization. Block fixation in aldehydes inhibits the enzyme activity (glutaraldehyde more than formaldehyde). The osmificated azo-dye originated of 4-methoxy-2-naphthylamine and Fast Blue B or hexazonium-p-rosaniline is still partially soluble in solvents used for the usual embedding in epoxyresins for electron microscopical examination. This is a drawback for a reliable demonstration of DAP IV in endothelial cells on the electronmicroscopical level using the epoxy-resin technique. DAP IV of the endothelium is inhibited totally by DFP (10^–3M), partially by E 600 (10^–3); and slightly by phenanthroline (10^–3M). It is unaffected by EDTA (10^–3M) and N-ethyl maleimide (10^–3M).The combined demonstration of alkaline phosphatase and DAP IV in the same section renders a reliable demonstration of the capillary bed in many organs.The contribution of DAP IV activity of the capillary endothelium to the total DAP IV activity in a particular organ is decisive in the myocardium, striated muscle, aorta and lung; it represents about one half of the total activity in spleen and pancreas and is less expressed in the liver, intestine and particularly in the kidney.In the jejunum of patients sufferring coeliac sprue the activity of capillary endothelium in the propria is decreased or not demonstrable in the acute stage. After a gluten-free diet it is restituted. The activity of DAP IV does not change significantly in aortae of the rabbit and man with atherosclerosis. In plaques of human aortae the capillary endothelium reacts at the most. Vasa vasorum in the adventitia overlying large plaques, which penetrate into the media, display a high DAP IV activity and their number can be increased. In plaques of arteries of cocks there is a positive DAP IV reaction in foam cells. DAP IV does not belong to the enzymes indicating early changes in atherogenesis.The function of DAP IV in the endothelium is not known. It may be a part of the machinery influeneing the protein part of the endothelial coat or may participate in the degradation of some vasoactive peptides.     

Further purification of "Triton subchloroplast fraction I" (TSF-I particles). Isolation of a cytochrome-free high-P-700 particle and a complex containing cytochromes f and b6, plastocyanin and iron-sulfur protein(s).

The "Triton Subchloroplast Fraction I" or "TSF-I particles" can be further fractionated into a cytochrome fraction and a P-700-containing fraction essentially free of cytochromes. The cytochrome complex contains cytochromes f and b6 in approx. equimolar amounts, and, in addition, also plastocyanin and one iron-sulfur protein, all in the bound state. Bound plastocyanin was characterized by EPR spectroscopy. The EPR spectrum of the bound iron-sulfur protein resembles that previously detected in Phostosystem I particles under highly reducing conditions at lower than -560 mV. The redox potential of P-700 in the cytochrome-free high-P-700 particles was measured to be +468 mV; those of cytochromes f and b6 are +345 and -140 mV, respectively. Among the four components present in the complex, only cytochrome f can be coupled to a Photosystem I particle and undergoes photooxidation. This coupled photooxidation is totoally inhibited by KCN and only partially inhibited by HgCl2. The similarity of the complex containing cytochromes f and b6, plastocyanin, and an iron-sulfur protein to complexes III and IV of the mitochondrial respiratory redox chain and a possible involvement of the complex in cyclic photophosphorylation are noted and discussed.     

Pseudomonas aeruginosa diaminopimelate decarboxylase: evolutionary relationship with other amino acid decarboxylases

The lysA gene encodes meso-diaminopimelate (DAP) decarboxylase (E.C.4.1.1.20), the last enzyme of the lysine biosynthetic pathway in bacteria. We have determined the nucleotide sequence of the lysA gene from Pseudomonas aeruginosa. Comparison of the deduced amino acid sequence of the lysA gene product revealed extensive similarity with the sequences of the functionally equivalent enzymes from Escherichia coli and Corynebacterium glutamicum. Even though both P. aeruginosa and E. coli are Gram-negative bacteria, sequence comparisons indicate a greater similarity between enzymes of P. aeruginosa and the Gram- positive bacterium C. glutamicum than between those of P. aeruginosa and E. coli enzymes. Comparison of DAP decarboxylase with protein sequences present in data bases revealed that bacterial DAP decarboxylases are homologous to mouse (Mus musculus) ornithine decarboxylase (E.C.4.1.1.17), the key enzyme in polyamine biosynthesis in mammals. On the other hand, no similarity was detected between DAP decarboxylases and other bacterial amino acid decarboxylases.      

Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides

Many chloroplast proteins are synthesized in the cytoplasm as precursors which contain an amino terminal transit peptide. These precursors are subsequently imported into chloroplast and targeted to one of several organellar locations. This import is mediated by the transit peptide, which is cleaved off during import. We have used the transit peptides of ferredoxin (chloroplast stroma) and plastocyanin (thylakoid lumen) to study chloroplast protein import and intra-organellar routing toward different compartments. Chimeric genes were constructed that encode precursor proteins in which the transit peptides are linked to yeast mitochondrial manganese superoxide dismutase. Chloroplast protein import and localization experiments show that both chimeric proteins are imported into the chloroplast stroma and processed. The plastocyanin transit sequence did not direct superoxide dismutase to the thylakoids; this protein was found in the stroma as an intermediate that still contains part of the plastocyanin transit peptide. The organelle specificity of these chimeric precursors reflected the transit peptide parts of the molecules, because neither the ferredoxin and plastocyanin precursors nor the chimeric proteins were imported into isolated yeast mitochondria.     

Cytochemical reactivity of T mu lymphocytes in human lymphatic tissue for dipeptidylaminopeptidase IV

Summary The enzyme dipeptidylaminopeptidase IV (DAP IV; EC 3.4.14.-) was recently shown cytochemically to be confined, in blood and bone marrow, to human T cells bearing, Fc receptors for IgM (T lymphocytes). This observation, confirmed by direct biochemical tests, stimulated us to study the histochemical distribution of DAP IV in normal human lymphatic tissue. In cryostat sections of lymph node, tonsil and thymus, DAP IV was detectable only in lymphocytes, Hassal's corpuscles and the endothelia of vessels. The distribution pattern of DAP IV-positive lymphocytes accorded well with results obtained with human T cell antisera. Compared to cytochemical reactions for other enzymes, such as acid esterase, DAP IV has the advantage that it does not stain monocytes, B lymphocytes or other mononuclear cells. Further, it does not depend on a particular type of staining pattern like, for example, the dot-like reaction product of acid esterase in T lymphocytes. Since the reaction for DAP IV remains more or less unchanged in month-old sections, it is easily adaptable to routine work and has the potentiality of being applied to the diagnosis of T cell lymphomas.     

Characterisation of human dipeptidyl peptidase IV expressed in Pichia pastoris. A structural and mechanistic comparison between the recombinant human and the purified porcine enzyme

Dipeptidyl peptidase IV/CD26 (DP IV) is a multifunctional serine protease cleaving off dipeptides from the N-terminus of peptides. The enzyme is expressed on the surface of epithelial and endothelial cells as a type II transmembrane protein. However, a soluble form of DP IV is also present in body fluids. Large scale expression of soluble human recombinant His(6)-37-766 DP IV, using the methylotrophic yeast Pichia pastoris, yielded 1.7 mg DP IV protein per litre of fermentation supernatant. The characterisation of recombinant DP IV confirmed proper folding and glycosylation similar to DP IV purified from porcine kidney. Kinetic comparison of both proteins using short synthetic substrates and inhibitors revealed similar characteristics. However, interaction analysis of both proteins with the gastrointestinal hormone GLP-1(7-36) resulted in significantly different binding constants for the human and the porcine enzyme (Kd = 153.0 +/- 17.0 microM and Kd = 33.4 +/- 2.2 microM, respectively). In contrast, the enzyme adenosine deaminase binds stronger to human than to porcine DP IV (Kd = 2.15 +/- 0.18 nM and Kd = 7.38 +/- 0.54 nM, respectively). Even though the sequence of porcine DP IV, amplified by RT-PCR, revealed 88% identity between both enzymes, the species-specific variations between amino acids 328 to 341 are likely to be responsible for the differences in ADA-binding.     

Serum activities of dipeptidyl-aminopeptidase II and dipeptidyl-aminopeptidase IV in tumor-bearing animals and in cancer patients

Serum activities of dipeptidyl-aminopeptidases (DAP) II and IV were measured in tumor-bearing animals and in patients with blood and solid cancers using highly sensitive and specific fluorometric methods. In mice with intraperitoneal or subcutaneous implantation of Ehrlich ascites tumor cells, serum DAP II activity was increased and serum DAP IV activity was decreased, resulting in a significant increase in the ratio of serum DAP II and DAP IV activities. The increase in the ratio of these two activities paralleled the size of the subcutaneous tumors. However, both serum DAP II and DAP IV activities were increased in rats with experimental hepatic cancer induced by 3'-methyl-4-dimethylaminoazobenzene, and the increase in the ratio of the two activities was not significant. In cancer patients, as compared with healthy subjects, serum DAP II activity was increased and serum DAP IV activity was decreased, the ratio of serum DAP II and DAP IV activities being markedly increased in cancer patients. Both serum DAP II and DAP IV activities were increased in patients with hepatic cancer as were those in rats with hepatic cancer, but the increase in DAP II was greater than that of DAP IV; thus the ratio of the two activities increased significantly. These data suggest that the increase of the serum DAP II/DAP IV ratio could be a biochemical index of cancer.     

Effects of natural shade on soybean thylakoid membrane composition

The effect of natural shade on chloroplast thylakoid membrane activity and composition was examined for soybean (Glycine Max. cv. Young) grown under field conditions. Plots with high (10 plants m^–1 row) or low (1 plant m^–1 row) plant density were established. Expanding leaves were tagged at 50, 58 and 65 days after planting (DAP). At 92 DAP, tagged leaves were used as reference points to characterize canopy light environments and isolate thylakoid membranes. Light environments ranged from a photosynthetic photon flux density (PPFD) of 87% of full sun to a PPFD of 10% of full sun. The decline in PPFD was accompanied by an increase in the far-red/red (735 nm/645 nm) ratio from 0.9 to approximately six. The major effects of shade on chloroplast thylakoid membranes were a reduction in chloroplast coupling factor and a shift in light-harvesting capacity from Photosystem I to Photosystem II. Photosynthetic electron transport capacity was not affected by differences in PPFD, but was 20 to 30% higher in the 1 plant m^–1 row treatment. The plant density effect on electron transport was associated with differences in plastocyanin concentration, suggesting that plastocyanin is a limiting factor in soybean. Shade did not have a significant effect on the concentration of Photosystem II, Cyt b₆f, or Photosystem I complexes.Abbreviations CF₁ chloroplast coupling factor - DAP days after planting - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCIP 2,6-dichlorophenolindophenol - FR/R far-red/red - PBS 10 mM sodium phosphate (pH 7.0), 150 mM NaCl - PPFD photosynthetic photon flux density - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TBS 20 mM Tris-HCl (pH 7.5), 500 mM NaCl - TTBS 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.05% (w/v) polyoxyethylenesorbitan monolaurate (Tween-20) The US Government right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.The US Government right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.     

Transient binding of plastocyanin to its physiological redox partners modifies the copper site geometry

The transient complexes of plastocyanin with cytochrome f and photosystem I are herein used as excellent model systems to investigate how the metal sites adapt to the changes in the protein matrix in transient complexes that are involved in redox reactions. Thus, both complexes from the cyanobacterium Nostoc sp. PCC 7119 (former Anabaena sp. PCC 7119) have been analysed by X-ray absorption spectroscopy. Our data are consistent with a significant distortion of the trigonal pyramidal geometry of the Cu coordination sphere when plastocyanin binds to cytochrome f, no matter their redox states are. The resulting tetrahedral geometry shows a shortening of the distance between Cu and the S(delta) atom of its ligand Met-97, with respect to the crystallographic structure of free plastocyanin. On the other hand, when plastocyanin binds to photosystem I instead of cytochrome f, the geometric changes are not significant but a displacement in charge distribution around the metal centre can be observed. Noteworthy, the electronic density around the Cu atom increases or decreases when oxidised plastocyanin binds to cytochrome f or photosystem I, respectively, thus indicating that the protein matrix affects the electron transfer between the two partners during their transient interaction.     

Mesentery vascular metabolism of substance P

Dipeptidylpeptidase IV (EC 3.4.14.5), an enzyme which metabolizes substance P, is present in crude homogenates of hog mesenteric artery and aorta. Its subcellular localization is closely correlated with the plasma membrane marker enzyme 5'-nucleotidase (EC 3.1.3.5) in addition to the kinin and angiotensin metabolizing enzymes angiotensin I converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). The highest level of dipeptidylpeptidase IV is found on the surface membrane-enriched fraction and is immunologically identical to the kidney brush border-bound enzyme. Vascular dipeptidylpeptidase IV sequentially removes the N-terminal Arg1-Pro2 and Lys3-Pro4 dipeptides of substance P and exposes the biologically active C-terminal heptapeptide product to rapid degradation by vascular aminopeptidases.     

Genetic variation in soybean photosynthetic electron transport capacity is related to plastocyanin concentration in the chloroplast

Fifteen ancestral genotypes of United States soybean cultivars were screened for differences in photosynthetic electron transport capacity using isolated thylakoid membranes. Plants were grown in controlled environment chambers under high or low irradiance conditions. Thylakoid membranes were isolated from mature leaves. Photosynthetic electron transport was assayed as uncoupled Hill activity using 2,6-dichlorophenolindophenol (DCIP). Soybean electron transport activity was dependent on genotype and growth irradiance and ranged from 6 to 91 mmol DCIP reduced [mol chlorophyll]^–1 s^–1. Soybean plastocyanin pool size ranged from 0.1 to 1.3 mol plastocyanin [mol Photosystem I]^–1. In contrast, barley and spinach electron transport activities were 140 and 170 mmol DCIP reduced [mol chlorophyll]^–1 s^–1, respectively, with plastocyanin pool sizes of 3 to 4 mol plastocyanin [mol Photosystem I]^–1. No significant differences in the concentrations of Photosystem II, plastoquinone, cytochrome b₆f complexes, or Photosystem I were observed. Thus, genetic differences in electron transport activity were correlated with plastocyanin pool size. The results suggested that plastocyanin pool size can vary significantly and may limit photosynthetic electron transport capacity in certain species such as soybean. Soybean plastocyanin consisted of two isoforms with apparent molecular masses of 14 and 11 kDa, whereas barley and spinach plastocyanins each consisted of single polypeptides of 8 and 12 kDa, respectively.Abbreviations DAP days after planting - DCIP 2,6-dichlorophenolindophenol - LiDS lithium dodecyl sulfate - PPFD photosynthetic photon flux density (mol photons m^–2 s^–1) - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Identification of the plastocyanin binding subunit of photosystem I

Plastocyanin is specifically cross-linked by incubation with N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) to a subunit of photosystem I in stroma lamellae and in isolated photosystem I complex. SDS-PAGE shows the disappearance of a 18.5 kDa subunit and the appearance of a new 31.5 kDa protein which was recognized by anti-plastocyanin antibodies. The isolated subunit was identified by its N-terminal amino acid sequence as the mature peptide coded by the nuclear gene psaF [Steppuhn et al. (1988) FEBS Lett. 237, 218–224]. P700⁺ was reduced by cross-linked plastocyanin with the same halftime of 13 μs as found in the native complex. This is evidence that cross-linking conserved the orientation of the complex and that the 18.5 kDa subunit provides the conformation of photosystem I necessary for the extremely rapid electron transfer from plastocyanin to P700⁺.     

Isolation and characterization of Chromatium vinosum membranes

A fractionation of Chromatium vinosum into an outer layer (cell wall) and three intracellular membrane fractions by isopycnic sucrose density centrifugation of a total membrane fraction obtained by lysis of lysozyme-EDTA spheroplasts is decribed. The three intracellular fractions (I, II, and III) have apparent densities of 1.11, 1.14, and 1.16, respectively, and contain the bulk of the photosynthetic pigments. Fraction II is enriched in bacteriochlorophyll and contains about 49% of the total membrane protein and 60% of the membrane bacteriochlorophyll. The outer membrane fraction (IV, cell wall) has a density of 1.23 and contains 5% of the membrane protein and 0.8% of the bacteriochlorophyll. Fraction I is enriched in lipids and phosphorus and has only a trace of diaminopimelate (DAP). Fractions II and III both contain a significant content of DAP. Fraction IV has no DAP, but has a fatty acid composition similar to that of the envelope fraction. Electrophoresis of the fractions on sodium dodecylsulfate-containing gels yielded from 8–13 bands of protein. Fractions I, II, and III contained the same series of unique proteins, while fraction IV contained another group of unique proteins. In fraction IV the bulk of the proteins traveled in one band with a molecular weight of 41,500. Examination of the fractions and whole spheroplasts in the electron microscope showed that fractions I, II and III were composed of large membrane structures in the form of membrane reticulum with bud-like appendages, and elongated flattened tubes. Fraction IV was composed of large ovoid structures which were seen to lie on the outer surface of the whole spheroplasts. These results suggest that the normal in vivo state of the intracellular membranes is that of an interconnected series of tubules and vesicles extending throughout the cell cytoplasm.     

S-Adenosyl-L-methionine-dependent restriction enzymes

Restriction-modification (R-M) enzymes are classified into type I, II, III, and IV, based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements. While the role of S-Adenosyl-L-methionine (AdoMet) as the methyl group donor in the methylation reaction is undisputed, its requirement in DNA cleavage reaction has been subject to intense study. AdoMet is a prerequisite for the DNA cleavage by most type I enzymes known so far, with the exception of R.EcoR124I. A number of new type II restriction enzymes belonging to the type IIB and IIG family were found to show AdoMet dependence for their cleavage reaction. The type III enzymes have been found to require AdoMet for their restriction function. AdoMet functions as an allosteric effector of the DNA cleavage reaction and has been shown to bring about conformational changes in the protein upon binding.