Acid phosphatase and adenosine triphosphatase activities in the cell wall of baker's yeast.

In order to establish whether a specific adenosine triphosphatase is present in yeast cell wall, hydrolysis rates for p-nitrophenylphosphate (acid phosphatase activity) and for ATP (ATPase activity) were compared under various conditions. Rate determinations were made with both, intact cells and with preparations containing secreted enzymes from protoplasts. Acid phosphatase and ATPase activities had the same pH profile and were susceptible in the same way to the repression by orthophosphate and to the inhibition by 2-deoxyglucose. The Lineweaver-Burk plot shows biphasic kinetic behaviour for the hydrolysis of either p-nitrophenylphosphate or ATP. This suggests the existence of two enzymes with different affinities for the substrates, or one enzyme with at least two active sites. The two activities differ in thermostability and only one activity could be completely abolished by heat treatment. The thermostable enzyme activity had K-m values of 0.475 mM for p-nitrophenylphosphate, and 0.040 mM for ATP. ATP behaved as a partially competitive inhibitor of p-nitrophenylphosphate hydrolysis. Substrate competition studies showed that only a non-specific acid phosphatase is responsible for the hydrolysis of ATP.     

Structure of supported bilayers composed of lipopolysaccharides and bacterial phospholipids: raft formation and implications for bacterial resistance

Lipopolysaccharide (LPS), the major lipid on the surface of Gram-negative bacteria, plays a key role in bacterial resistance to hydrophobic antibiotics and antimicrobial peptides. Using atomic force microscopy (AFM) we characterized supported bilayers composed of LPSs from two bacterial chemotypes with different sensitivities to such antibiotics and peptides. Rd LPS, from more sensitive "deep rough" mutants, contains only an inner saccharide core, whereas Ra LPS, from "rough" mutants, contains a longer polysaccharide region. A vesicle fusion technique was used to deposit LPS onto either freshly cleaved mica or polyethylenimine-coated mica substrates. The thickness of the supported bilayers measured with contact-mode AFM was 7 nm for Rd LPS and 9 nm for Ra LPS, consistent with previous x-ray diffraction measurements. In water the Ra LPS bilayer surface was more disordered than Rd LPS bilayers, likely due to the greater volume occupied by the longer Ra LPS polysaccharide region. Since deep rough mutants contain bacterial phospholipid (BPL) as well as LPS on their surfaces, we also investigated the organization of Rd LPS/BPL bilayers. Differential scanning calorimetry and x-ray diffraction indicated that incorporation of BPL reduced the phase transition temperature, enthalpy, and average bilayer thickness of Rd LPS. For Rd LPS/BPL mixtures, AFM showed irregularly shaped regions thinner than Rd LPS bilayers by 2 nm (the difference in thickness between Rd LPS and BPL bilayers), whose area increased with increasing BPL concentration. We argue that the increased permeability of deep rough mutants is due to structural modifications caused by BPL to the LPS membrane, in LPS hydrocarbon chain packing and in the formation of BPL-enriched microdomains.     

Peptidyl - tRNA hydrolase and RNase activities in cell fractions of rat liver used in in vitro reconstitution of rough membrane.

Peptidyl-tRNA hydrolase and RNase activities have been studied in those fractions of rat liver, which are used in in vitro reconstitution of rough membrane, because these enzymes may interfere with the in vitro reconstitution. It was found that smooth membrane has an active peptidyl-tRNA hydrolase, while the other fractions tested, polyribosomes, rough membrane, stripped rough membrane and the post-microsomal supernatant had no, or very low, peptidyl-tRNA hydrolase activity. Polyribosomes, rough and stripped rough membrane have RNase activity; this activity could be completely inhibited by rat liver RNase inhibitor. It is shown that RNase inhibitor is an obligatory component in in vitro experiments, in which rough membrane is reconstituted from stripped rough membrane, ribosomes and mRNA.     

Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of gamma-glutamyl kinase.

A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.     

Gloverins of the silkworm Bombyx mori: Structural and binding properties and activities

Gloverins are basic, glycine-rich and heat-stable antibacterial proteins (～14- kDa) in lepidopteran insects with activity against Escherichia coli, Gram-positive bacteria, fungi and a virus. Hyalophora gloveri gloverin adopts a random coil structure in aqueous solution but has α-helical structure in membrane-like environment, and it may interact with the lipid A moiety of lipopolysaccharide (LPS). Manduca sexta gloverin binds to the O-specific antigen and outer core carbohydrate of LPS. In the silkworm Bombyx mori, there are four gloverins with slightly acidic to neutral isoelectric points. In this study, we investigate structural and binding properties and activities of B. mori gloverins (BmGlvs), as well as correlations between structure, binding property and activity. Recombinant BmGlv1-4 were expressed in bacteria and purified. Circular dichroism (CD) spectra showed that all four BmGlvs mainly adopted random coli structure (>50%) in aqueous solution in regardless of pH, but contained α-helical structure in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), smooth and rough mutants (Ra, Rc and Re) of LPS and lipid A. Plate ELISA assay showed that BmGlvs at pH 5.0 bound to rough mutants of LPS and lipid A but not to smooth LPS. Antibacterial activity assay showed that positively charged BmGlvs (at pH 5.0) were active against E. coli mutant strains containing rough LPS but inactive against E. coli with smooth LPS. Our results suggest that binding to rough LPS is the prerequisite for the activity of BmGlvs against E. coli.     

Hydrolysis of 13-sophorosyloxydocosanoic acid esters by acetyl- and carboxylesterases isolated from Candida bororiensis.

Two enzymes have been isolated from Candida bogoriensis which catalyze the hydrolysis of 13-sophorosyloxydocosanoic acid (Glc2HDA) esters obtained from this organism. The 6',6"-diacetyl derivative of Glc2HDA (Ac2Glc2HDA) is hydrolyzed by an acetylesterase (EC 3.1.1.6) which has been purified 1300-fold. The acetylesterase has a molecular weight of 35,000 estimated from gel filtration, and is much more active with p-nitrophenyl acetate than with the acetylated glycolipid. The rate of hydrolysis increases with Ac2Glc2HDA concentration until a plateau is reached at a concentration of about 40 muM, near the critical micelle concentration of this glycolipid. These kinetic data are interpreted as an enzyme specificity for the monomeric, but not the micellar form of the glycolipid. The acetylesterase is inhibited by 0.1 to 10 mM diisopropyl fluorophosphate, 5 mM p-hydroxymercuribenzoate, and 5 mM N-ethylmaleimide, but only slightly by 5 mM iodoacetamide. The methyl ester of Ac2Glc2HDA is hydrolyzed by at least two carboxylesterases (EC 3.1.1) which differ in size according to gel filtration. Their molecular weights are estimated as 140,000 for carboxyesterase A and 40,000 for carboxyesterase B. Both carboxylesterases were purified over 20-fold, and carboxylesterase A was characterized further. Carboxylesterase A activity was inhibited completely by 0.1 to 10 mM diisopropyl fluorophosphate and by 10 mM p-hydroxymercuribenzoate, but only slightly by lower concentrations of p-hydroxymercuribenzoate or by N-ethylmaleimide or iodoacetamide. The carboxylesterase A preparation also acted as a thioesterase with palmityl-CoA (palmityl-CoA hydrolase, EC 3.1.2.2), showing the following approximate relative activities: palmityl-CoA, 100; octanoyl-CoA, 90; methyl Glc2HD, 22; butyryl-CoA, 18; methyl AcGlc2HD, 15; methyl Ac2Glc2HD, 10; and acetyl-CoA, O. Methyl Ac2Glc2HD showed some substrate inhibition at higher concentrations, but neither methyl Ac2Glc2HD nor palmityl-CoA approached enzyme saturation until well above their critical micelle concentrations, indicating hydrolysis of the micellar substrate was occurring. The carboxylesterase and palmityl-CoA hydrolase activities were destroyed in a parallel fashion by heat denaturation, and each substrate inhibited the action of the preparation on the other substrate, but the preparation has not been purified sufficiently to establish with certainty that both activities reside in the same protein.     

Characterization of polyagglutinating and surface antigens in Pseudomonas aeruginosa

Mutants of Pseudomonas aeruginosa PAC1R (serotype O:3) which were resistant to bacteriophage D were isolated and shown to react with O:5d, O:9 and O:13 antisera as well as O:3. Antisera to the parent strain and to the three polyagglutinating (PA) mutants also showed cross-reactions. The mutants differed from the parent strain in their lipopolysaccharide (LPS) composition. The LPS from two of the three mutants yielded high molecular weight polysaccharide fractions. Although the high molecular weight fraction from one of the mutants contained the amino sugars and other components characteristic of the O:3 serotype strains, its mobility on Sephadex G75 was different from that of the parent strain. The high molecular weight material from the second mutant lacked the O-antigenic determinants but these were present in a semi-rough LPS fraction. The third mutant appeared rough and completely lacked the O-antigenic components. These three mutants were compared with the parent strain and with a non-agglutinating LPS-defective mutant which lacked both O-antigenic side chains and all neutral sugars in the outer core. Agglutination with absorbed sera and haemagglutination using purified LPS and ELISA detection suggested that wall components other than LPS were responsible for some of the cross-reactions observed. The components responsible were detected after SDS-PAGE of crude outer membrane fractions by a combination of Coomassie blue and silver-staining and antigenic components were detected by immunoelectrophoresis and ELISA-linked immunoblotting of the gels. The main antigenic determinants detected by antiserum to the parent strain were in the high molecular weight O-polysaccharide fractions and in the semirough fractions of the LPS, with some activity due to the H protein of the outer membrane. O:5d antisera reacted with unidentified high molecular weight polysaccharide fractions. Cross-reactions with the O:9 antiserum appeared to be due mainly to the F porin and, to a lesser extent, to the G and E proteins of the outer membrane. O:13 antiserum reacted with high molecular weight polysaccharide fractions but also with the rough core and F and H protein. Cross-reactivity of the other three mutant antisera could largely be interpreted in terms of the outer membrane components exposed in each strain. One reacted strongly with the F porin and the rough core, while the others reacted with a number of protein and LPS-derived fractions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane structure of the human immunodeficiency virus gp41 fusion peptide by molecular dynamics simulation. II. The glycine mutants

In this work, molecular dynamics (MD) simulation of the interaction of three mutants, G3V, G5V and G10V, of the human immunodeficiency virus (HIV) gp41 16-residue fusion peptide (FP) with an explicit palmitoyloleoylphosphatidyl-ethanolamine (POPE) lipid bilayer was performed. The goals of this work are to study the correlation of the fusogenic activity of the FPs with the mode of their interaction with the bilayer and to examine the roles of the many glycine residues in the FP in the fusion process. The results of this work corroborate the main conclusion of our earlier MD work of the WT FP and several mutants with polar substitution. These two studies provide correlation between the mode of insertion and the fusogenic activity of these peptides and support the hypothesis that an oblique insertion of the fusion domain of the viral protein is required for fusogenic activity. Inactive mutants interact with the bilayer by a surface-binding mode. The results of this work, combined with the results of our earlier work, show that, while the secondary structures of the wild-type FP and its mutants do not affect the fusogenic activities, the conformational flexibility appears to be an important factor. The active WT FP and its partially active mutants, G3V and G5V, all have significant conformational transitions at one of the glycine sites. They occur at Gly(5) in FP-wt, at Gly(10) in FP-G5V and at Gly(13) in FP-G3V. Thus, a glycine site in each of these active (or partially active) FPs provides conformational flexibility. On the other hand, the inactive mutants FP-G10V, FP-L9R and FP-V2E do not have any conformational transitions except at either terminus and thus possess no conformational flexibility. Thus, the results of this work support the suggestion that the role of glycine residues in the fusion domain is to provide the necessary conformational flexibility for fusion activity.The glycines also form a "glycine strip" in the FP that locates on one (the less hydrophobic) face of the helix (the "sided helix"). However, whether this "glycine strip" is disrupted or not does not seem to correlate with the retention of fusogenic activities. Finally, although the FLGFL (8-12) motif is absolutely conserved in the HIV fusion domain, a well-structured motif stabilized by hydrogen bonding does not appear to be required for activity. In fact, hydrogen bonding in this motif was found to be missing in FP-G3V and FP-G5V. Both of these mutants are partially active.     

Human bisphosphoglycerate mutase. Expression in Escherichia coli and use of site-directed mutagenesis in the evaluation of the role of the carboxyl-terminal region in the enzymatic mechanism

Bisphosphoglycerate mutase is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate, the allosteric effector of hemoglobin. In addition to its main 2,3-diphosphoglycerate synthase activity, the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. The three activities have been demonstrated to be catalysed at a unique active site. To study the structure of such an active site we have developed a recombinant system producing mutants of human bisphosphoglycerate mutase in Escherichia coli, by site-directed mutagenesis. For this purpose the human bisphosphoglycerate mutase cDNA that we had previously cloned has been used to construct a procaryotic high level expression vector bearing the "tac" promoter. Human bisphosphoglycerate mutase produced in E. coli, a species which does not normally synthesize this enzyme, represented 8% of the total soluble bacterial protein and displayed the three catalytic activities (synthase, mutase, and phosphatase) characteristic of the enzyme. Since it has been suggested that the carboxyl-terminal region may be implicated in the catalytic activity of the enzyme, three variants deleted in this part of the protein were produced. Our results indicate that a minimal deletion of 7 amino acid residues in the carboxyl-terminal portion of the human bisphosphoglycerate mutase completely abolished the three catalytic activities of the enzyme. In contrast, the effects of the deletion of the last two lysine residues were limited to a 38% reduction in the synthase activity. These results show that the carboxyl-terminal amino acid residues are either directly or indirectly implicated in the three catalytic functions of the human bisphosphoglycerate mutase, and that the two terminal lysine residues are not essential for the major part of the enzymatic mechanism of the enzyme.     

A single residue at the active site of CD38 determines its NAD cyclizing and hydrolyzing activities

CD38 is a multifunctional enzyme involved in metabolizing two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When incubated with NAD, CD38 predominantly hydrolyzes it to ADP-ribose (NAD glycohydrolase), but a trace amount of cADPR is also produced through cyclization of the substrate. Site-directed mutagenesis was used to investigate the amino acid important for controlling the hydrolysis and cyclization reactions. CD38 and its mutants were produced in yeast, purified, and characterized by immunoblot. Glu-146 is a conserved residue present in the active site of CD38. Its replacement with Phe greatly enhanced the cyclization activity to a level similar to that of the NAD hydrolysis activity. A series of additional replacements was made at the Glu-146 position including Ala, Asn, Gly, Asp, and Leu. All the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly. E146A showed the highest cyclase activity, which was more than 3-fold higher than its hydrolysis activity. All mutants also cyclized nicotinamide guanine dinucleotide to produce cyclic GDP. This activity was enhanced likewise, with E146A showing more than 9-fold higher activity than the wild type. In addition to NAD, CD38 also hydrolyzed cADPR effectively, and this activity was correspondingly depressed in the mutants. When all the mutants were considered, the two cyclase activities and the two hydrolase activities were correlated linearly. The Glu-146 replacements, however, only minimally affected the base-exchange activity that is responsible for synthesizing NAADP. Homology modeling was used to assess possible structural changes at the active site of E146A. These results are consistent with Glu-146 being crucial in controlling specifically and selectively the cyclase and hydrolase activities of CD38.     

Bacterial lipopolysaccharides as inducers of disease resistance in tobacco.

The cell wall component of Pseudomonas solanacearum that induces disease resistance in tobacco was highly heat stable at neutral or alkaline pH but highly labile at acid pH. Activity was unaffected by nucleases and proteases but destroyed by a mixture of beta-glycosidases. Washing of bacterial cell walls released a lipopolysaccharide (LPS) fraction with high inducer activity. Purified LPS, extracted by a variety of procedures from whole cells, isolated cell walls, and culture filtrates of both smooth and rough forms of P. solanacearum, induced disease resistance in tobacco at concentrations as low as 50 microgram/ml. The LPS from the non-plant pathogens Escherichia coli B, E. coli K, and Serratia marcescens was also active. Cell wall protein, free phospholipid, and nucleic acids were not necessary for activity. Moreover, since LPS from rough forms was active, the O-specific polysaccharide of the LPS was not required for activity. Hydrolysis of the remaining core-lipid A linkage or deacylation of lipid A destroyed inducer activity. When injected into tobacco leaves, purified LPS attached to tobacco mesophyll cell walls and induced ultrastructural changes in the host cell similar to those induced by attachment of whole heat-killed bacteria.     

Bacterial lipopolysaccharides as inducers of disease resistance in tobacco.

The cell wall component of Pseudomonas solanacearum that induces disease resistance in tobacco was highly heat stable at neutral or alkaline pH but highly labile at acid pH. Activity was unaffected by nucleases and proteases but destroyed by a mixture of beta-glycosidases. Washing of bacterial cell walls released a lipopolysaccharide (LPS) fraction with high inducer activity. Purified LPS, extracted by a variety of procedures from whole cells, isolated cell walls, and culture filtrates of both smooth and rough forms of P. solanacearum, induced disease resistance in tobacco at concentrations as low as 50 microgram/ml. The LPS from the non-plant pathogens Escherichia coli B, E. coli K, and Serratia marcescens was also active. Cell wall protein, free phospholipid, and nucleic acids were not necessary for activity. Moreover, since LPS from rough forms was active, the O-specific polysaccharide of the LPS was not required for activity. Hydrolysis of the remaining core-lipid A linkage or deacylation of lipid A destroyed inducer activity. When injected into tobacco leaves, purified LPS attached to tobacco mesophyll cell walls and induced ultrastructural changes in the host cell similar to those induced by attachment of whole heat-killed bacteria.     

Isolation and characterization of bacteriophage FC3-10 from Klebsiella spp.

FC3-10 is a Klebsiella spp. specific bacteriophage isolated on a rough mutant (strain KT707, chemotype Rd) of K. pneumoniae C3. The bacteriophage receptor for this phage was shown to be the low-molecular mass lipopolysaccharide (LPS) fraction (LPS-core oligosaccharides), specifically the heptose content of the LPS inner-core. This is the first phage isolated on Klebsiella, the receptor for which is the LPS-core. This phage was unable to plate on Salmonella typhimurium LPS mutants with chemotypes Rd2 or Re showing incomplete or no heptose content on their LPS-core, respectively. Spontaneous phage-resistant mutants from different Klebsiella strains were deep-rough LPS mutants or encapsulated revertants from unencapsulated mutant strains.     

Lipid A, the active part of bacterial endotoxins in inducing serum colony stimulating activity and proliferation of splenic granulocyte/macrophage progenitor cells.

An analysis of which component of lipopolysaccharides (LPS), the lipid or the polysaccharide (PS), is active in stimulating the murine granulopoietic system has been performed. LPS with different structures, isolated from different mutant strains of Salmonella and chemical degradation products of lipopolysaccharides have been used. Lipid A obtained by acid hydrolysys of the LPS and complexed to bovine serum albumin (BSA) (lipid A-BSA) was shown to be active in generating serum colony stimulating factor (CSF) and in increasing the splenic colony forming cells (CFC) levels, although it was less active than the parent LPS. The polysaccharide (PS) showed no significant activity at the concentrations used. LPS (glycolipids) from R mutants of Salmonella minnesota were active to the same extent as the LPS. The fact that even the most defective LPS from the R mutant R595 which contains lipid A and KDO only is a potent endotoxin, points unequivocally, to lipid A, as the active principle in stimulating the granulopoietic system.     

The extraction and analysis of lipopolysaccharides from Pseudomonas aeruginosa strain PAO, and three rough mutants.

Three spontaneously arising rough mutants of Pseudomonas aeruginosa have been isolated by selection for resistance to virulent lipopolysaccharide (LPS) specific bacteriophages. In addition, the first phages specific for rough mutants of P. aeruginosa were isolated. Using these phage and autoagglutination patterns in 4% NaCl and acriflavine, these mutants could be clearly distinguished from the wild-type strain and each other. Chemical analysis of the LPS together with chromatographic resolution of the polysaccharide moieties showed alterations in both O-specific side chains and core regions.     

Formate hydrogenlyase system in Salmonella typhimurium LT2.

Isolation from Salmonella typhimurium of mutants unable to reduce benzyl viologen under anaerobic conditions has allowed the study of the factors involved in the multienzymic formate hydrogenylase system. 1. Depending on the affected activities, different classes of mutants were found: FHL-A mutants have lost formate dehydrogenase 1 and formate dehydrogenase 2 activities; mutations in fdhA (117 min) or fdhB (33 min) lead to such a phenotype. FHL-B and FHL-C mutants have lost formate dehydrogenase 2 activity and part or all of hydrogenase activity, respectively; both types correspond to mutations in the hyd gene (approximately 90 min). FHL-D mutants have lost only formate dehydrogenase 2 activity; fhlD gene maps at 120 min. 2. In some cases, mixtures of extracts from two mutants display formate dehydrogenase 2 and formate hydrogenylase activities. Restoration studies suggest the existence of one factor sensitive to growth conditions and inactivated by oxygen or heating. This factor which is present and active in FHL-C mutants, is probably the one missing in FHL-D mutants. 3. A new scheme for the formate hydrogenylase system is proposed, in which hydrogenase transfers electrons directly to benzyl viologen.     

Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution.

The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1 (serotype O5) and IATS O6 (serotype O6), generated isogenic mutants from them, and examined the distribution of LPS on the surface of these organisms by freeze-substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31- to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype O6 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A- B-), rd7513 (A- B-), and R5 (an IATS O6-derived rough mutant; A- B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that freeze-substitution electron microscopy is effective in the visualization of LPS morphotypes.     

Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase.

We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases.