Probing a functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase through transesterification reactions in organic solvent

To reveal the functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased theV _max,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. TheK _m,app for tributyrin was not affected by the Glu87Ala mutation, but theK _m,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreasedV _max,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on theK _m,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased theK _m,app for tributyrin and vinyl butyrate by a factor of >5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0–680 mM) studied here. With vinyl butyrate as a substrate theV _max,app was only 6% of that obtained with wild-type enzyme.     

Synthesis and SAR of thieno[3,2-b]pyridinyl urea derivatives as urotensin-II receptor antagonists

The preparation and SAR profile of thieno[3,2-b]pyridinyl urea derivatives as novel and potent urotensin-II receptor antagonists are described. An activity optimization study, probing the effects of substituents on thieno[3,2-b]pyridinyl core and benzyl group of the piperidinyl moiety, led to the identification of p-fluorobenzyl substituted thieno[3,2-b]pyridinyl urea 6n as a highly potent UT antagonist with an IC₅₀ value of 13 nM. Although 6n displays good metabolic stability and low hERG binding activity, it has an unacceptable oral bioavailability.     

Structural and functional studies of a trans‐acyltransferase polyketide assembly line enzyme that catalyzes stereoselective α‐ and β‐ketoreduction

While the cis‐acyltransferase modular polyketide synthase assembly lines have largely been structurally dissected, enzymes from within the recently discovered trans‐acyltransferase polyketide synthase assembly lines are just starting to be observed crystallographically. Here we examine the ketoreductase (KR) from the first polyketide synthase module of the bacillaene nonribosomal peptide synthetase/polyketide synthase at 2.35‐Å resolution. This KR naturally reduces both α‐ and β‐keto groups and is the only KR known to do so during the biosynthesis of a polyketide. The isolated KR not only reduced an N‐acetylcysteamine‐bound β‐keto substrate to a D ‐β‐hydroxy product, but also an N‐acetylcysteamine‐bound α‐keto substrate to an L ‐α‐hydroxy product. That the substrates must enter the active site from opposite directions to generate these stereochemistries suggests that the acyl‐phosphopantetheine moiety is capable of accessing very different conformations despite being anchored to a serine residue of a docked acyl carrier protein. The features enabling stereocontrolled α‐ketoreduction may not be extensive since a KR that naturally reduces a β‐keto group within a cis‐acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same α‐keto substrate to generate the D ‐α‐hydroxy product. A sequence analysis of trans‐acyltransferase KRs reveals that a single residue, rather than a three‐residue motif found in cis‐acyltransferase KRs, is predictive of the orientation of the resulting β‐hydroxyl group. Proteins 2014; 82:2067–2077. © 2014 Wiley Periodicals, Inc.     

Design,synthesis and evaluation against Mycobacterium tuberculosis of azole piperazine derivatives as dicyclotyrosine (cYY) mimics

Three series of azole piperazine derivatives that mimic dicyclotyrosine (cYY), the natural substrate of the essential Mycobacterium tuberculosis cytochrome P450 CYP121A1, were prepared and evaluated for binding affinity and inhibitory activity (MIC) against M. tuberculosis. Series A replaces one phenol group of cYY with a C3-imidazole moiety, series B includes a keto group on the hydrocarbon chain preceding the series A imidazole, whilst series C explores replacing the keto group of the piperidone ring of cYY with a CH₂-imidazole or CH₂-triazole moiety to enhance binding interaction with the heme of CYP121A1. The series displayed moderate to weak type II binding affinity for CYP121A1, with the exception of series B 10a, which displayed mixed type I binding. Of the three series, series C imidazole derivatives showed the best, although modest, inhibitory activity against M. tuberculosis (17d MIC?=?12.5?μg/mL, 17a 50?μg/mL). Crystal structures were determined for CYP121A1 bound to series A compounds 6a and 6b that show the imidazole groups positioned directly above the haem iron with binding between the haem iron and imidazole nitrogen of both compounds at a distance of 2.2?Å. A model generated from a 1.5?Å crystal structure of CYP121A1 in complex with compound 10a showed different binding modes in agreement with the heterogeneous binding observed. Although the crystal structures of 6a and 6b would indicate binding with CYP121A1, the binding assays themselves did not allow confirmation of CYP121A1 as the target.     

CF3‐Pyridinyl Substitution on Antimalarial Therapeutics: Probing Differential Ligand Binding and Dynamical Inhibitory Effects of a Novel Triazolopyrimidine‐Based Inhibitor on Plasmodium falciparum Dihydroorotate Dehydrogenase

The quest for reliable dihydroorotate dehydrogenase (DHODH) inhibitors has engendered the discovery of potential therapeutic compounds at different stages of clinical trials. Although promising, high attrition rates and unfavorable bioactivities have limited their drug developmental progress. A recent structural modification of DSM265, a triazolopyrimidine‐based inhibitor, yielded DSM421, derived by the substitution of the SF₅‐aniline group on DSM265 with a CF₃‐pyridinyl moiety. Consequently, DSM421 exhibited improved pharmacological and pharmacokinetics attributes relative to DSM265. The improved bioactivity mediated by the CF₃‐pyridinyl group leaves us with a curiosity to investigate underlying ligand‐binding mechanisms and dynamics using computational methods. Presented in this study are insights that clearly explain the effects of structural SF₅‐aniline→CF₃‐pyridinyl modifications on pfDHODH inhibition. Findings showed that the CF₃‐pyridinyl group induced an optimal and stabilized positioning of DSM421 within the binding pocket, allowing for steady and strong intermolecular interactions which favored its stronger binding affinity as estimated and correlated with bioactivity data. These interactions consequently induced a pronounced stabilization of the structural conformation of pfDHODH by restricting residue motions, which possibly underpinned its enhanced inhibitory activity relative to DSM265. Active site interactions of the CF₃‐pyrinidyl group with residues Ser236, Ile237, and Phe188 characterized by strong π–π stacking and halogen interactions also stabilized its positioning which altogether accounted for its enhanced inhibitory prowess towards pfDHODH. On the contrary, fewer and weaker interactions characterized DSM265 binding which could explain its relatively lower binding affinity. Findings will facilitate the design of novel pfDHODH inhibitors with enhanced properties.     

Computer Modelling Studies on the Mechanism of Action of Ribonuclease T1

Abstract

The mechanism of action of ribonuclease (RNase) T₁ is still a matter of considerable debate as the results of x-ray, 2-D nmr and site-directed mutagenesis studies disagree regarding the role of the catalytically important residues. Hence computer modelling studies were carried out by energy minimisation of the complexes of RNase T₁ and some of its mutants (His40Ala, His40Lys, and Glu58Ala) with the substrate guanyl cytosine (GpC), and of native RNase T₁ with the reaction intermediate guanosine 2′, 3′-cyclic phosphate (G>p). The puckering of the guanosine ribose moiety in the minimum energy conformer of the RNase T₁ - GpC (substrate) complex was found to be O4′-endo and not C3′-endo as in the RNase T₁ - 3′-guanylic acid (inhibitor/product) complex. A possible scheme for the mechanism of action of RNase T₁ has been proposed on the basis of the arrangement of the catalytically important amino acid residues His40, Glu58, Arg77, and His92 around the guanosine ribose and the phosphate moiety in the RNase T₁ - GpC and RNase T₁ - G>p complexes. In this scheme, Glu58 serves as the general base group and His92 as the general acid group in the transphosphorylation step. His40 may be essential for stabilising the negatively charged phosphate moiety in the enzyme-transition state complex.     

Structural and functional investigation of AerF,a NADPH-dependent alkenal double bond reductase participating in the biosynthesis of Choi moiety of aeruginosin

The 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety is an essential residue for the antithrombotic activities of aeruginosins, which are a class of cyanobacterial derived bioactive linear tetrapeptides. Biosynthetic pathway of Choi is still elusive. AerF was suggested to be involved in the biosynthesis of Choi, and can be assigned to the short-chain dehydrogenase/reductase (SDR) superfamily. However, both the exact role and the catalytic mechanism of AerF have not been elucidated. In this study, functional and mechanistic analyses of AerF from Microcystis aeruginosa were performed. Observation of enzymatic assay demonstrates that AerF is a NADPH-dependent alkenal double bond reductase that catalyzes the reduction of dihydro-4-hydroxyphenylpyruvate (H₂HPP) to generate tetrahydro-4-hydroxyphenylpyruvate (H₄HPP), which is the third step of the biosynthetic pathway from prephenate to Choi. Comparative structural analysis indicates that ligand binding-induced conformational change of AerF is different from that of the other SDR superfamily reductase using H₂HPP as a substrate. Analyses of NADPH and substrate analogue binding sites combined with the results of mutagenesis analyses suggest that a particular serine residue mainly involves in the initiation of the proton transfer between the substrate and the residues of AerF, which is an uncommon feature in SDR superfamily reductase. Furthermore, based on the observations of structural and mutagenesis analyses, the catalytic mechanism of AerF is proposed and a proton transfer pathway in AerF is deduced.     

Changing the target base specificity of the EcoRV DNA methyltransferase by rational de novo protein-design

The EcoRV DNA-(adenine-N⁶)-methyltransferase (M.EcoRV) specifically modifies the first adenine residue within GATATC sequences. During catalysis, the enzyme flips its target base out of the DNA helix and binds it into a target base binding pocket which is formed in part by Lys16 and Tyr196. A cytosine residue is accepted by wild-type M.EcoRV as a substrate at a 31-fold reduced efficiency with respect to the k_cat/K_M values if it is located in a CT mismatch substrate (GCTATC/GATATC). Cytosine residues positioned in a CG base pair (GCTATC/GATAGC) are modified at much more reduced rates, because flipping out the target base is much more difficult in this case. We intended to change the target base specificity of M.EcoRV from adenine-N⁶ to cytosine-N⁴. To this end we generated, purified and characterized 15 variants of the enzyme, containing single, double and triple amino acid exchanges following different design approaches. One concept was to reduce the size of the target base binding pocket by site-directed mutagenesis. The K16R variant showed an altered specificity, with a 22-fold preference for cytosine as the target base in a mismatch substrate. This corresponds to a 680-fold change in specificity, which was accompanied by only a small loss in catalytic activity with the cytosine substrate. The K16R/Y196W variant no longer methylated adenine residues at all and its activity towards cytosine was reduced only 17-fold. Therefore, we have changed the target base specificity of M.EcoRV from adenine to cytosine by rational protein design. Because there are no natural paragons for the variants described here, a change of the target base specificity of a DNA interacting enzyme was possible by rational de novo design of its active site.     

Primary structural requirements for N- and O-glycosylation of yeast mannoproteins

Primary structural requirements both for N- and O-glycosylation have been studied using a series of synthetic peptides and a membrane fraction from Saccharomyces cerevisiae. N-Glycosylation: the tripeptide sequence Asn-Xaa-Thr/Ser was found to be necessary for the transfer of saccharide units from oligosaccharide-lipid to asparagine. Substitution of asparagine by aspartic acid or glutamine, or replacement of threonine by valine in the hexapeptide Tyr-$\text{Asn}$-Leu-$\text{Thr}$-Ser-Val prevents its glycosylation. Also, a proline residue in the position of Xaa makes the peptide unable to function as an acceptor. Transfer onto asparagine occurs only efficiently if both the α-amino group of asparagine and the α-carboxyl moiety of the hydroxy amino acid are blocked. Yield of glycosylation improves with increasing peptide chain length. With regard to the glycosyl donor dolichyl diphosphate-bound GlcNAc₂Man₉Glc₃ is the preferred substrate. Non-glucosylated glycolipid Dol-PP-GlcNAc₂Man₉ is a poor donor, whereas smaller precursors Dol-PP-GlcNAc₂ and Dol-PP-GlcNAc₂Man₁ allow reasonable transfer. O-Glycosylation: no marker sequence can be derived for the formation of an O-glycosidic linkage via Dol-P-Man. Introduction of a proline residue in vicinity to the hydroxy amino acid leads to a significant improvement of glycosyl transfer. It is postulated that accessibility of potential O-glycosylation sites rather than a specific sequence may be a prerequisite for O-glycosylation.     

The role of a conserved tyrosine in the 49-kDa subunit of complex I for ubiquinone binding and reduction

Iron–sulfur cluster N2 of complex I (proton pumping NADH:quinone oxidoreductase) is the immediate electron donor to ubiquinone. At a distance of only ～ 7 Å in the 49-kDa subunit, a highly conserved tyrosine is found at the bottom of the previously characterized quinone binding pocket. To get insight into the function of this residue, we have exchanged it for six different amino acids in complex I from Yarrowia lipolytica. Mitochondrial membranes from all six mutants contained fully assembled complex I that exhibited very low dNADH:ubiquinone oxidoreductase activities with n-decylubiquinone. With the most conservative exchange Y144F, no alteration in the electron paramagnetic resonance spectra of complex I was detectable. Remarkably, high dNADH:ubiquinone oxidoreductase activities were observed with ubiquinones Q₁ and Q₂ that were coupled to proton pumping. Apparent K_m values for Q₁ and Q₂ were markedly increased and we found pronounced resistance to the complex I inhibitors decyl-quinazoline-amine (DQA) and rotenone. We conclude that Y144 directly binds the head group of ubiquinone, most likely via a hydrogen bond between the aromatic hydroxyl and the ubiquinone carbonyl. This places the substrate in an ideal distance to its electron donor iron–sulfur cluster N2 for efficient electron transfer during the catalytic cycle of complex I.     

Downregulation of TLR4 and 7 mRNA Expression Levels in Broiler’s Spleen Caused by Diets Supplemented with Nickel Chloride

Toll-like receptors (TLRs) are important immune receptors in discriminating self from nonself and in initiating the innate and adaptive immune response. TLR4 and TLR7 have been proven to be highly expressed in chicken’s spleen. Thus, this study was to evaluate the TLR4 and TLR7 messenger RNA (mRNA) expression levels in the spleen of broilers fed diets supplemented with nickel chloride (NiCl₂) using the methods of quantitative real-time PCR (qRT-PCR). Two hundred forty-one-day-old avian broilers were equally divided into 4 groups and fed on a corn-soybean basal diet as control diet or the same basal diet supplemented with 300, 600, and 900 mg/kg of NiCl₂ for 42 days. Results showed that TLR4 and TLR7 mRNA expression levels in the spleen were lower (P?<?0.05 or P?<?0.01) in the 300, 600, and 900 mg/kg groups than those in the control group. It was concluded that dietary NiCl₂ in excess of 300 mg/kg could lower TLR4 and TLR7 mRNA expression levels in the spleen of broilers, implying that NiCl₂ could impair the innate and adaptive immunity in spleen by injuring immunocytes and/or decreasing the content of cytokines through TLRs.     

Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit

Substitution of the queuine nucleobase precursor preQ₁ by an azide-containing derivative (azido-propyl-preQ₁) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNA^Asp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a ‘minimally invasive’ system for placement of a non-natural, clickable nucleobase within the total cellular RNA.     

Molecular modeling approach to predict a binding mode for the complex methotrexate-carboxypeptidase G<Subscript>2</Subscript>

Carboxypeptidase G₂ (CPG₂) is a zinc-metalloenzyme employed in a range of cancer chemotherapy strategies by activating selectively nontoxic prodrugs into cytotoxic drugs in tumor as well as in the treatment of intoxication caused by high-doses of the anticancer drug methotrexate (MTX). CPG₂ catalyzes the hydrolytic cleavage of C-terminal of glutamate moiety from folic acid and analogues. Regardless of its extensive application, its mechanism of catalysis has not yet been determined and, so far, no co-crystallized complex has been published. So, in this study, molecular docking and a short molecular dynamics (MD) simulation sampling scheme, as a function of temperature, were performed to investigate a possible binding mode for MTX, a recognized substrate of CPG₂. The findings suggested that MTX interacts possibly in quite specific points of the CPG₂ active site, which are probably responsible for the molecular recognition and cleavage procedures. The MTX substrate fits well in the catalytic site by accommodating the pteridine moiety in an adjacent pocket to the active site whereas a glutamate moiety is pointed toward the protein surface. Additionally, a glutamate residue can interact with a crystallization water molecule in the active site, supporting its activation as a nucleophilic group.     

Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met→Ala168 mutant penicillin acylases from Kluyvera citrophila

The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters K_m and k_cat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met→Ala168 mutation unstabilizes the first transition-state (E··S^≠) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site.     

Amino acid substrate specificity of asparaginyl-,aspartyl- and glutaminyl-tRNA synthetase isolated from higher plants

AspNH₂-, Asp- and GluNH₂-tRNA synthetases were purified from Phaseolus aureus; their optimum assay conditions, substrate specificities and salt sensitivities were investigated. AspNH₂-tRNA synthetase from β-cyanoalanine-producing (Vicia sativa), and non-producing (P. aureus and V. faba) species was able to utilize the analogue as a substrate irrespective of the source of the enzyme. Asp-tRNA synthetase from P. aureus was able to utilize α-aminomalonate and threo-β-hydroxy Asp as a substrate. The transfer of ¹⁴C-GluNH₂ to tRNA, catalyzed by GluNH₂-tRNA synthetase, was only inhibited by high concentrations of those analogues tested; albizziine was the most efficient, but no difference could be demonstrated between the substrate specificities of the enzyme isolated from an albizziine-producer (A. julibrissin and a non-producer (P. aureus) species.     

Autocatalytic Cleavage within Classical Swine Fever Virus NS3 Leads to a Functional Separation of Protease and Helicase

Classical swine fever virus (CSFV) is a positive-stranded RNA virus belonging to the genus Pestivirus within the Flaviviridae family. Pivotal for processing of a large portion of the viral polyprotein is a serine protease activity within nonstructural protein 3 (NS3) that also harbors helicase and NTPase activities essential for RNA replication. In CSFV-infected cells, NS3 appears as two forms, a fully processed NS3 of 80 kDa and the precursor molecule NS2-3 of 120 kDa. Here we report the identification and mapping of additional autocatalytic intramolecular cleavages. One cleavable peptide bond occurs between Leu₁₇₈₁ and Met₁₇₈₂, giving rise to a helicase subunit of 55 kDa and, depending on the substrate, a NS2-3 fragment of 78 kDa (NS2-3p) or a NS3 protease subunit of 26 kDa (NS3p). In trans-cleavage assays using NS4-5 as a substrate, NS3p acts as a fully functional protease that is able to process the polyprotein. NS3p comprises the minimal essential protease, as deletion of Leu₁₇₈₁ results in inactivation. A second intramolecular cleavage was mapped to the Leu₁₇₄₈/Lys₁₇₄₉ peptide bond that yields a proteolytically inactive NS3 fragment. Deletion of either of the cleavage site residues resulted in a loss of RNA infectivity, indicating the functional importance of amino acid identity at the respective positions. Our data suggest that internal cleavage within the NS3 moiety is a common process that further extends the functional repertoires of the multifunctional NS2-3 or NS3 and represents another level of the complex polyprotein processing of Flaviviridae.     

Structural basis for catalysis by onconase

Onconase® (ONC) is a homolog of bovine pancreatic ribonuclease (RNase A) from the frog Rana pipiens. ONC displays antitumoral activity and is in advanced clinical trials for the treatment of cancer. Here, we report the first atomic structures of ONC-nucleic acid complexes: a T89N/E91A ONC-5′-AMP complex at 1.65 Å resolution and a wild-type ONC-d(AUGA) complex at 1.90 Å resolution. The latter structure and site-directed mutagenesis were used to reveal the atomic basis for substrate recognition and turnover by ONC. The residues in ONC that are proximal to the scissile phosphodiester bond (His10, Lys31, and His97) and uracil nucleobase (Thr35, Asp67, and Phe98) are conserved from RNase A and serve to generate a similar bell-shaped pH versus k_cat/K_M profile for RNA cleavage. Glu91 of ONC forms two hydrogen bonds with the guanine nucleobase in d(AUGA), and Thr89 is in close proximity to that nucleobase. Installing a neutral or cationic residue at position 91 or an asparagine residue at position 89 virtually eliminated the 10²-fold guanine:adenine preference of ONC. A variant that combined such substitutions, T89N/E91A ONC, actually preferred adenine over guanine. In contrast, installing an arginine residue at position 91 increased the guanine preference and afforded an ONC variant with the highest known k_cat/K_M value. These data indicate that ONC discriminates between guanine and adenine by using Coulombic interactions and a network of hydrogen bonds. The structure of the ONC-d(AUGA) complex was also used to probe other aspects of catalysis. For example, the T5R substitution, designed to create a favorable Coulombic interaction between ONC and a phosphoryl group in RNA, increased ribonucleolytic activity by twofold. No variant, however, was more toxic to human cancer cells than wild-type ONC. Together, these findings provide a cynosure for understanding catalysis of RNA cleavage in a system of high medicinal relevance.     

Development of substrate analogue inhibitors for the human airway trypsin-like protease HAT

A series of substrate analogue inhibitors of the serine protease HAT, containing a 4-amidinobenzylamide moiety as the P1 residue, was prepared. The most potent compounds possess a basic amino acid in the d-configuration as P3 residue. Whereas inhibitor 4 (K_i 13 nM) containing proline as the P2 residue completely lacks selectivity, incorporation of norvaline leads to a potent inhibitor (15, K_i 15 nM) with improved selectivity for HAT in comparison to the coagulation proteases thrombin and factor Xa or the fibrinolytic plasmin. Selected inhibitors were able to suppress influenza virus replication in a HAT-expressing MDCK cell model.     

Methylated nucleotide content of mitochondrial ribosomal RNA from hamster cells

We have examined the state of methylation of mitochondrial ribosomal RNA from cultured hamster (BHK-21) cells. Ethidium-sensitive, and hence mitochondrion-specific, methylation levels were determined using multiple isotope techniques and improved purification procedures. The larger mitochondrial rRNA species, 17 S RNA, was found to contain 0.13 methyl group per 100 nucleotides and the smaller, 13 S RNA, 0.37. Methylated nucleotide and base analysis indicated that 17 S RNA contained one ribose-methylated residue (UmUp) per molecule and one unidentified residue; and 13 S RNA contained one methylated cytosine residue, one N⁶-dimethyladenine residue and one thymine residue per molecule. Possible evolutionary implications of these findings have been discussed.