Modification of tomato andAspergillus niger pectinesterases with diethyl pyrocarbonate

The role of histidine residues in pectinesterases was evaluated by monitoring the sensitivity to modification with diethyl pyrocarbonate in the tomato andAspergillus niger enzymes. Different and incomplete losses of enzyme activity were obtained. Inactivation of the enzymes was proportional to the histidine content (two in the tomato T1 form, six in theAspergillus form), suggesting that accessible histidine residues do not have active-site functions in these pectinesterases, but contribute to the overall structural stability. Lack of His roles in common between the enzyme forms is in agreement with the structures of pectinesterases having no conserved His residues.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Chemical Modification of Catalytically Essential Functional Groups of NAD-Dependent Hydrogenase from Ralstonia eutropha H16

Amino acid residues His and Cys of the NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha H16 were chemically modified with specific reagents. The modification of His residues of the nonactivated hydrogenase resulted in decrease in both hydrogenase and diaphorase activities of the enzyme. Activation of NADH hydrogenase under anaerobic conditions additionally modified a His residue (or residues) significant only for the hydrogenase activity. The rate of decrease in the diaphorase activity was unchanged. The modification of thiol groups of the nonactivated enzyme did not affect the hydrogenase activity. The effect of thiol-modifying agents on the activated hydrogenase was accompanied by inactivation of both diaphorase and hydrogenase activities. The modification degree and changes in the corresponding catalytic activities depended on conditions of the enzyme activation. Data on the modification of cysteine and histidine residues of the hydrogenase suggested that the enzyme activation should be associated with significant conformational changes in the protein globule.     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co²⁺, Hg²⁺, Ag⁺, Mg²⁺ and Ca²⁺ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol^–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.     

Evidence that three histidine residues of a base non-specific and adenylic acid preferential ribonuclease from Rhizopus niveus are involved in the catalytic function.

In order to study the structure-function relationship of an RNase T2 family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or His109 showed less than 0.03%, but a mutant enzyme at His104 showed 0.54% of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or His109 towards 2'-AMP decreased twofold, but that at His104 decreased more markedly. Therefore, we assumed that these three histidine residues are components of the active site of RNase Rh, that His104 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, His104, and His109 by comparison of the 1H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual pKa values for His46 and His104 to be 6.70 and 5.94. His109 was not titrated in a regular way, but the apparent pKa value was estimated to be around 6.3. The fact that addition of 2'-AMP caused a greater effect on the chemical shift of His104 in the 1NMR spectra as compared with those of the other histidine residues, may support the idea described above on the role of His104.     

A histidine decarboxylase-like mRNA is involved in tomato fruit ripening

DNA sequencing of a tomato ripening-related cDNA, TOM 92, revealed an open reading frame with homology to several pyridoxal 5-phosphate histidine decarboxylases, containing the conserved amino acid residues known to bind pyridoxal phosphate and -fluoromethylhistidine, an inhibitor of enzyme activity. TOM 92 mRNA accumulated during early fruit ripening and then declined. Fruit of the ripeningimpaired tomato mutant, ripening inhibitor (rin), did not accumulate TOM 92 mRNA, and its accumulation was not restored by treatment of fruit with ethylene. The TOM 92 mRNA was not detected in tomato leaves and unripe fruit.     

Functional Expression of <Emphasis Type="Italic">Spirulina</Emphasis>-Δ<Superscript>6</Superscript> Desaturase Gene in Yeast, <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Spirulina-acyl-lipid desaturases are membrane-bound enzymes found in thylakoid and plasma membranes. These enzymes carry out the fatty acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. In this study, Spirulina-Δ⁶ desaturase encoded by the desD gene was heterologously expressed and characterized in Saccharomyces cerevisiae. We then conducted site-directed mutagenesis of the histidine residues in the three histidine boxes to determine the role of these amino acid residues in the enzyme function. Our results showed that while four mutants showed complete loss of Δ⁶-desaturase activity and two mutants showed only trace of the activity, the enzyme activity could be partially restored by chemical rescue using exogenously provided imidazole. This study reveals that the histidine residues (which have imidazole as their functional group) in the conserved clusters play a critical role in Δ⁶-desaturase activity, possibly by providing a di-iron catalytic center. In our previous study, this enzyme was expressed in Escherichia coli. The results reveal that the enzyme can function only in the presence of an exogenous cofactor, ferredoxin, provided in vitro. This evidence suggests that baker’s yeast has a cofactor that can complement ferredoxin, thought to act as an electron donor for the Δ⁶ desaturation in cyanobacteria, including Spirulina. The electron donor of the Spirulina-Δ⁶ desaturation in yeast is more likely to be cytochrome b₅, which is absent in E. coli. This means that the enzyme expressed in S. cerevisiae can catalyze the biosynthesis of the product, GLA, in vivo.     

Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli.

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.     

Proton magnetic resonance studies on Escherichia coli dihydrofolate reductase. Assignment of histidine C-2 protons in binary complexes with folates on the basis of the crystal structure with methotrexate and on chemical modifications.

The effects of pH upon the C-2 resonances of the 5 histidine residues of Escherichia coli MB 1428 dihydrofolate reductase in binary complexes with methotrexate, aminopterin, folate, methopterin, and trimethoprim were studied by 300-MHz 1H nmr spectroscopy. Three of the five histidine residues, labeled 1, 2, and 3, exhibited similar pK' values and chemical shifts for their C-2 protons in the five binary complexes. One histidine, 4, was quite different in the folate complex and the last histidine, 5 was quite different in the trimethoprim complex. For all five binary complexes, each histidine had a pK' which was significantly different from the other 4 histidines of that complex. Titration of the binary methotrexate complex of a 5,5'-dithiobis(2-nitrobenzoate)-modified enzyme showed that 2 histidines were not perturbed by this modification of Cys 152, and that the alkaline form of histidine 2, the acid form of histidine 4, and, to a lesser extent, the acid form of histidine 3 were slightly perturbed. Titration of the binary methotrexate complex of a N-bromosuccinimide-modified enzyme demonstrated that this modification slightly affected all of the histidines and drastically affected histidine 5. Histidines 3 and 5 of the binary methotrexate complex reacted rapidly with the histidine-specific reagent, ethoxyformic anhydride, while histidines 2 and 4 reacted at a moderate rate and histidine 1 reacted slowly if at all. The local electrostatic environments of the 5 histidine residues as deduced from the crystal structure of the binary complex of the enzyme with methotrexate (Matthews, D.A., Alden, R.A., Bolin, J.T., Freer, S.T., Hamlin, R., Xuong, N., Kraut, J., Poe, M., Williams, M.N., and Hoogsteen, K. (1977) Science 197, 594-597) were used as the basis for proposed assignments of the five histidine C-2 nmr resonances. The assignments were: 1, pK' 7.9 to 8.2, His 124; 2, pK' 7.2 to 7.4, His 141; 3, pK' 6.5 to 6.7, His 149; 4, pK' 5.7 to 6.3, His 114; and 5, pK' 5.2 to 5.9, His 45. The effect of the chemical modifications upon the enzyme's histidine residues were consistent with the assignments, but no direct chemical evidence in support of the assignments was obtained. It was proposed that, since the crystallographic data provided consistent assignments of the histidine nmr data for both native and chemically modified enzyme, the local environment of each of the 5 histidine residues was similar in the crystal and in solution.     

Identification of histidine residues important in the catalysis and structure of aspartyl aminopeptidase

Aspartyl aminopeptidase (DAP), a widely distributed and abundant cytosolic enzyme, removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. DAP is a member of the M18 family of the MH clan of cocatalytic metallopeptidases. The human and mouse enzymes have been cloned. We have identified 8 highly homologous eukaryotic sequences that are probable aspartyl aminopeptidases. Eight histidine residues of human DAP were sequentially mutated to phenylalanine. Mutation of His94, His170, and His440 abolished enzymatic activity. His94 and His440 are postulated to be involved in binding cocatalytic zinc atoms by homology with other members of the MH clan. Mutation of His352 dramatically reduced enzyme activity. Gel-filtration analysis of the His352 mutant revealed destabilization of the quaternary structure and dissociation of the native 440-kDa enzyme. Mutation of His33 and of histidines residing in a cluster at residues 349, 359, and 363 all decreased k(cat). These studies reveal an important role for histidine residues both in catalysis and in the structural integrity of DAP.     

Site-directed mutagenesis of the conserved histidine residue of phosphoenolpyruvate carboxylase. His138 is essential for the second partial reaction.

Histidine residues have previously been suggested to be essential for the activity of phosphoenolpyruvate carboxylase as demonstrated by chemical modification of these residues. Although the location of these residues on the primary structure is not known, a comparison of nine phosphoenolpyruvate (P-pyruvate) carboxylases sequenced recently revealed that there are only two conserved histidine residues (His138 and His579, coordinates from the E. coli enzyme). Site-directed mutagenesis of these residues were undertaken with the E. coli P-pyruvate carboxylase and the properties of purified mutant enzymes were investigated. Mutation of His138 to asparagine (H138N) produced a protein which did not show carboxylase activity. However, this mutant enzyme catalyzed the bicarbonate-dependent dephosphorylation (Vmax = 1.4 mumol.min-1.mg-1) of the P-pyruvate. Since this reaction is due to one of the two partial reactions proposed for this enzyme, the results indicate that His138 is obligatory for the second-step reaction, i.e. the carboxylation of the enolate form of pyruvate by carboxyphosphate. Mutation of His579 to asparagine (H579N) produced an enzyme which had 69% of the wild-type carboxylase activity, but its affinity for P-pyruvate was decreased by 24-fold.     

Disulfide bridges in tomato pectinesterase: variations from pectinesterases of other species; conservation of possible active site segments.

Analysis of tomato pectinesterase by carboxymethylation, with and without reduction, shows that the enzyme has two intrachain disulfide bridges. Analysis of fragments obtained from the native enzyme after digestion with pepsin identified bridges connecting Cys-98 with Cys-125, and Cys-166 with Cys-200. The locations of disulfide bridges in tomato pectinesterase are not identical to those in three distantly related pectinesterases (18-33% residue identities) from microorganisms. However, one half-Cys (i.e., Cys-166) position is conserved in all four enzymes. Sequence comparisons of the overall structures suggest a special importance for three short segments of the entire protein. One segment is at the N-terminal part of the tomato pectinesterase, another in the C-terminal portion near the distal end of the second disulfide loop, and the third segment is located in the central part between the two disulfide bridges. The latter segment, encompassing only 40 residues of the entire protein, appears to high-light a functional site in a midchain segment.     

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.     

Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus

Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn²⁺ that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K_m and dramatic decrease of k_cat, and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.     

Analysis of conserved glutamate residues in Porphyromonas gingivalis outer membrane receptor HmuR: toward a further understanding of heme uptake

The aim of this study was to broaden the current knowledge about the Porphyromonas gingivalis heme receptor HmuR. Site-directed mutagenesis was employed to replace Glu427, Glu448, Glu458 and Glu503 by alanines and to construct a triple Glu427Ala/Glu448Ala/Glu 458Ala mutant. All iron/heme-starved P. gingivalis mutants showed decreased growth recovery when human serum as the iron/heme source was used, hmuR::ermF, hmuR ^E503A and hmuR ^{E427A,E448A,E458A} mutant strains being the most affected. E. coli cells expressing HmuR with mutated glutamate residues bound hemin, hemoglobin and hemin–serum albumin complex with the same efficiency as did the wild-type recombinant protein, suggesting that the residues were not directly involved in heme binding. These data indicate that in addition to two conserved histidine residues (His95 and His434), NPDL and YRAP motifs, conserved glutamate residues are important for HmuR to utilize heme present in serum hemoproteins.     

Effects of the residue adjacent to the reactive serine on the substrate interactions ofDrosophila esterase 6

Esterase 6 fromDrosophila melanogaster is a carboxylesterase that belongs to the serine esterase multigene family. It has a basic histidine (His) at residue 187, adjacent to the reactive serine (Ser) at residue 188, whereas most other characterized members of the family have an acidic glutamate (Glu) in the equivalent position. We have used site-directedin vitro mutagenesis to replace the His codon of the esterase 6 gene with either Gln or Glu codons. The enzymes encoded by these active-site mutants and a wild-type control have been expressed, purified, and characterized. Substitution of Gln for His at position 187 has little effect on the biochemical properties of esterase 6, but the presence of Glu at this position is associated with three major differences. First, the pH optimum is increased from 7 to 9. Second, the mutant enzyme shows decreased activity for β-naphthyl esters andp-nitrophenyl acetate but has gained the ability to hydrolyze acetylthiocholine. Finally, the Gibb’s free energy of activation for the enzyme is increased. These results suggest that residue 187 interacts directly with the substrate alkyl group and that this interaction is fully realized in the transition state. We further propose that the presence of His rather than Glu at position 187 in esterase 6 contributes significantly to its functional divergence from the cholinesterases and that this divergence is due to different interactions between residue 187 and the substrate alkyl group.     

Probing the catalytically essential residues of a recombinant dipeptidyl carboxypeptidase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

In studying the structure and function of Escherichia coli dipeptidyl carboxypeptidase (EcDCP), we have employed in vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme in order to gain insight into the catalytic mechanism. Comparison of the amino acid sequence of EcDCP with other homologues indicates that the active site of the enzyme exhibits an HEXXH motif, a common feature of zinc metalloenzymes. The third metal binding ligand, presumed to coordinate directly to the active-site zinc ion in concert with His470 and His474 has been proposed as Glu499. Alterations to these residues completely abolished the catalytic activity against N-benzoyl-l-glycyl-l-histidyl-l-leucine. A significant loss of the enzymatic activity was also observed in F472V and F500V mutant enzymes. Intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in all mutant enzymes, whereas circular dichroism spectra were nearly identical for the tested proteins. Computer modeling suggests that residues His470, Glu471, His474, Glu499, and Phe500 are essential for EcDCP in maintaining the stable active-site environment. Taken together, these studies contribute to a more comprehensive understanding of the catalytic mechanism of the enzyme.     

Are the histidine residues of glutathione S-transferase important in catalysis? An assessment by 13C NMR spectroscopy and site-specific mutagenesis

To test the proposition that a histidine residue is essential in the catalytic mechanism of glutathione S-transferase, rat liver isoenzyme 3-3 specifically labeled with [ring-2-13C]histidine was prepared. The 13C NMR spectrum of the labeled enzyme revealed four resonances corresponding to the 4 histidine residues in the mu gene class type 3 subunit. Titration of the four resonances in the range of pH 4-9 both in the presence and absence of glutathione gave pK alpha values of much less than 4, 5.2, 7.1, and 7.8 for the four side chains that were identified by site-specific mutagenesis as His14, His83, His84, and His167, respectively. The magnetic resonance properties and titration behavior of His14 suggest that this residue is buried in a hydrophobic environment. Conservative replacement of each histidine with asparagine results in mutant enzymes that have catalytic properties very close to the native protein as assessed with three different substrates, 1-chloro-2,4-dinitrobenzene, 4-phenyl-3-buten-2-one, and phenanthrene 9,10-oxide. The results indicate clearly that none of the histidine residues of isoenzyme 3-3 is essential for stabilization of the bound glutathione thiolate or for any other aspect of catalysis.     

Identification of functionally relevant histidine residues in the apoptotic nuclease CAD

The caspase-activated DNase CAD (DFF40/CPAN) degrades chromosomal DNA during apoptosis. Chemical modification with DEPC inactivates the enzyme, suggesting that histidine residues play a decisive role in the catalytic mechanism of this nuclease. Sequence alignment of murine CAD with four homologous apoptotic nucleases reveals four completely (His242, His263, His304 and His308) and two partially (His127 and His313) conserved histidine residues in the catalytic domain of the enzyme. We have changed these residues to asparagine and characterised the variant enzymes with respect to their DNA cleavage activity, structural integrity and oligomeric state. All variants show a decrease in activity compared to the wild-type nuclease as measured by a plasmid DNA cleavage assay. H242N, H263N and H313N exhibit DNA cleavage activities below 5% and H308N displays a drastically altered DNA cleavage pattern compared to wild-type CAD. Whereas all variants but one have the same secondary structure composition and oligomeric state, H242N does not, suggesting that His242 has an important structural role. On the basis of these results, possible roles for His127, His263, His304, His308 and His313 in DNA binding and cleavage are discussed for murine CAD.