Extragenic suppression of the requirement for diaminopimelate in diaminopimelate auxotrophs of Mycobacterium smegmatis

Mycobacteria, like many prokaryotes, have a peptidoglycan with peptides composed of L-alanine (or glycine), D-iso-glutamine, meso-diaminopimelate, and D-alanine. We sought to study mycobacterial peptidoglycan biosynthesis by constructing diaminopimelate (DAP) auxotrophs of Mycobacterium smegmatis and then isolating spontaneous mutants of these auxotrophs that can grow in the absence of DAP. Here we report the isolation and characterization of seven classes of spontaneous M. smegmatis mutants with extragenic mutations that can suppress the DAP requirement of DAP auxotrophs.     

Unique structural features of the peptidoglycan of Mycobacterium leprae

The peptidoglycan structure of Mycobacterium spp. has been investigated primarily with the readily cultivable Mycobacterium smegmatis and Mycobacterium tuberculosis and has been shown to contain unusual features, including the occurrence of N-glycolylated, in addition to N-acetylated, muramic acid residues and direct cross-linkage between meso-diaminopimelic acid residues. Based on results from earlier studies, peptidoglycan from in vivo-derived noncultivable Mycobacterium leprae was assumed to possess the basic structural features of peptidoglycans from other mycobacteria, other than the reported replacement of l-alanine by glycine in the peptide side chains. In the present study, we have analyzed the structure of M. leprae peptidoglycan in detail by combined liquid chromatography and mass spectrometry. In contrast to earlier reports, and to the peptidoglycans in M. tuberculosis and M. smegmatis, the muramic acid residues of M. leprae peptidoglycan are exclusively N acetylated. The un-cross-linked peptide side chains of M. leprae consist of tetra- and tripeptides, some of which contain additional glycine residues. Based on these findings and genome comparisons, it can be concluded that the massive genome decay in M. leprae does not markedly affect the peptidoglycan biosynthesis pathway, with the exception of the nonfunctional namH gene responsible for N-glycolylmuramic acid biosynthesis.     

Decreased outer membrane permeability protects mycobacteria from killing by ubiquitin-derived peptides

Ubiquitin-derived peptides are bactericidal in vitro and contribute to the mycobactericidal activity of the lysosome. To further define interactions of ubiquitin-derived peptides with mycobacteria, we screened for mutants with increased resistance to the bactericidal activity of the synthetic ubiquitin-derived peptide Ub2. The four Ub2-resistant Mycobacterium smegmatis mutants were also resistant to the bactericidal action of other antimicrobial peptides and macrophages. Two mutants were in the mspA gene encoding the main M. smegmatis porin. Using a translocation-deficient MspA point mutant, we showed that susceptibility of M. smegmatis to Ub2 was independent of MspA channel activity. Instead, the M. smegmatis Ub2-resistant mutants shared a common phenotype of decreased cell wall permeability compared with wild-type bacteria. Expression of mspA rendered Mycobacterium tuberculosis CDC1551 more susceptible both to ubiquitin-derived peptides in vitro and to lysosomal killing in macrophages. Finally, biochemical assays designed to assess membrane integrity indicated that Ub2 treatment impairs membrane function of M. smegmatis and M. tuberculosis cells . The M. smegmatis Ub2-resistant mutants were more resistant than wild-type M. smegmatis to this damage. We conclude that Ub2 targets mycobacterial membranes and that reduced membrane permeability provides mycobacteria intrinsic resistance against antimicrobial compounds including bactericidal ubiquitin-derived peptides.     

Characterization of novel Mycobacterium tuberculosis and Mycobacterium smegmatis mutants hypersusceptible to beta-lactam antibiotics

Our laboratory previously constructed mutants of Mycobacterium tuberculosis and Mycobacterium smegmatis with deletions in the genes for their major beta-lactamases, BlaC and BlaS, respectively, and showed that the mutants have increased susceptibilities to most beta-lactam antibiotics, particularly the penicillins. However, there is still a basal level of resistance in the mutants to certain penicillins, and the susceptibilities of the mutants to some cephalosporin-based beta-lactams are essentially the same as those of the wild types. We hypothesized that characterizing additional mutants (derived from beta-lactamase deletion mutants) that are hypersusceptible to beta-lactam antibiotics might reveal novel genes involved with other mechanisms of beta-lactam resistance, peptidoglycan assembly, and cell envelope physiology. We report here the isolation and characterization of nine beta-lactam antibiotic-hypersusceptible transposon mutants, two of which have insertions in genes known to be involved with peptidoglycan biosynthesis (ponA2 and dapB); the other seven mutants have insertions which affect novel genes. These genes can be classified into three groups: those involved with peptidoglycan biosynthesis, cell division, and other cell envelope processes. Two of the peptidoglycan-biosynthetic genes (ponA2 and pbpX) may encode beta-lactam antibiotic-resistant enzymes proposed to be involved with the synthesis of the unusual diaminopimelyl linkages within the mycobacterial peptidoglycan.     

Expression, essentiality, and a microtiter plate assay for mycobacterial GlmU, the bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase

UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor of peptidoglycan and the rhamnose-GlcNAc linker region of mycobacterial cell wall. In Mycobacterium tuberculosis H37Rv genome, Rv1018c shows strong homology to the GlmU protein involved in the formation of UDP-GlcNAc from other bacteria. GlmU is a bifunctional enzyme that catalyzes two sequential steps in UDP-GlcNAc biosynthesis. Glucosamine-1-phosphate acetyl transferase catalyzes the formation of N-acetylglucosamine-1-phosphate, and N-acetylglucosamine-1-phosphate uridylyltransferase catalyzes the formation of UDP-GlcNAc. Since inhibition of peptidoglycan synthesis often results in cell lysis, M. tuberculosis GlmU is a potential anti-tuberculosis (TB) drug target. In this study we cloned M. tuberculosis Rv1018c (glmU gene) and expressed soluble GlmU protein in E. coli BL21(DE3). Enzymatic assays showed that M. tuberculosis GlmU protein exhibits both glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridylyltransferase activities. We also investigated the effect on Mycobacterium smegmatis when the activity of GlmU is fully removed or reduced via a genetic approach. The results showed that activity of GlmU is required for growth of M. smegmatis as the bacteria did not grow in the absence of active GlmU enzyme. As the amount of functional GlmU enzyme was gradually reduced in a temperature shift experiment, the M. smegmatis cells became non-viable and their morphology changed from a normal rod shape to stubby-rounded morphology and in some cases they lysed. Finally a microtiter plate based assay for GlmU activity with an OD340 read out was developed. These studies therefore support the further development of M. tuberculosis GlmU enzyme as a target for new anti-tuberculosis drugs.     

Species variation in ATP-dependent protein degradation: protease profiles differ between mycobacteria and protease functions differ between Mycobacterium smegmatis and Escherichia coli

We report here that the existence of the potentially broad substrate specificity protease Lon (also called La), is evolutionarily discontinuous within the order Actinomycetales. Lon homologues were identified in the fast-growing species Mycobacterium smegmatis, and the slow-growing species Micobacterium avium and Mycobacterium intracellulare. However, Lon homologues were not detected in the slow-growing species Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium leprae; or in the non-mycobacterial Actinomycetale Corynebacterium glutamica. To characterize the function of the Lon protease within the Actinomycetales, a viable M. smegmatis Deltalon strain was constructed, demonstrating that lon is not essential under certain conditions. Surprisingly, lon was also dispensable in M. smegmatis cells already lacking intact 20S proteasome alpha- and beta-subunit genes (called prcA and prcB, respectively). Creation of the later double deletion strain (prcBA::kan Deltalon) necessitated use of a novel gene deletion strategy that does not require an antibiotic resistance marker. The M. smegmatis prcBA::kan Deltalon double mutants displayed wild type (wt) growth rates and wt stress tolerances. In addition, the M. smegmatis prcBA::kan Deltalon double mutants degraded at wt rates the broad spectrum of truncated proteins induced by treating cells with puromycin. This later result was in sharp contrast to those in Escherichia coli, where either lon or hslUV single mutants are strongly impaired in their degradation of puromycyl peptides (hslV is a prcB homologue). Overall these data suggested that mycobacterial species contain additional ATP-dependent proteases that have broad substrate specificity. Consistent with this suggestion, M. smegmatis and M. tuberculosis each contain at least one homologue of ClpP, the proteolytic subunit common to the ClpAP and ClpXP proteases.     

A soluble enzyme activity that attaches free diaminopimelic acid to bdelloplast peptidoglycan

An enzyme activity, responsible for the attachment of diaminopimelic acid (DAP) to bdelloplast wall peptidoglycan, was studied in an in vitro, cell-free system. Most of the activity was found in the high-speed (20000g) supernatant fraction of homogenates of bdelloplasts prepared from a culture of the intracellular bacterium Bdellovibrio bacteriovorus 109J, growing synchronously within cells of Escherichia coli. Peptidoglycan preparations obtained either from E. coli ML35 or from the walls of bdelloplasts synchronously cultured for 40 or 90 min served as the acceptors in this reaction, whereas cell wall or peptidoglycan preparations obtained from Gram-positive bacteria could not function as acceptors of DAP. The attachment activity had an apparent Km value for DAP of 10 microM; for bdelloplast peptidoglycan, it was approximately 0.43 mg/mL, which is 13 microM with respect to peptidoglycan disaccharide peptide units. DAP attachment was partially inhibited by the structural analogues lanthionine, L-ornithine, beta-aminobutyric acid, and D-serine, as well as the cell wall synthesis inhibitors penicillin G, ampicillin, and cephalexin. This enzyme activity is present only during the intracellular stage of the bdellovibrio's developmental growth cycle and may serve a stage-specific function of biochemically modifying the cell in which it grows.     

Identification of a peptide synthetase involved in the biosynthesis of glycopeptidolipids of Mycobacterium smegmatis

Five rough colony mutants of Mycobacterium smegmatis mc2155 were produced by transposon mutagenesis. The mutants were unable to synthesize glycopeptidolipids that are normally abundant in the cell wall of wild-type M. smegmatis. The glycopeptidolipids have a lipopeptide core comprising a fatty acid amide linked to a tetrapeptide that is modified with O-methylated rhamnose and O-acylated 6-deoxy talose. Compositional analysis of lipids extracted from the mutants indicated that the defect in glycopeptidolipid synthesis occurred in the assembly of the lipopeptide core. No other defects or compensatory changes in cell wall structure were detected in the mutants. All five mutants had transposon insertions in a gene encoding an enzyme belonging to the peptide synthetase family. Targeted disruption of the gene in the wild-type strain gave a phenotype identical to that of the five transposon mutants. The M. smegmatis peptide synthetase gene is predicted to encode four modules that each contain domains for cofactor binding and for amino acid recognition and adenylation. Three modules also have amino acid racemase domains. These data suggest that the common lipopeptide core of these important cell wall glycolipids is synthesized by a peptide synthetase.     

Engineering a disulfide bond and free thiols in the lantibiotic nisin Z.

The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol-dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.     

Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange

Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.     

Role of peptidoglycan amidases in the development and morphology of the division septum in Escherichia coli

Escherichia coli contains multiple peptidoglycan-specific hydrolases, but their physiological purposes are poorly understood. Several mutants lacking combinations of hydrolases grow as chains of unseparated cells, indicating that these enzymes help cleave the septum to separate daughter cells after cell division. Here, we confirm previous observations that in the absence of two or more amidases, thickened and dark bands, which we term septal peptidoglycan (SP) rings, appear at division sites in isolated sacculi. The formation of SP rings depends on active cell division, and they apparently represent a cell division structure that accumulates because septal synthesis and hydrolysis are uncoupled. Even though septal constriction was incomplete, SP rings exhibited two properties of mature cell poles: they behaved as though composed of inert peptidoglycan, and they attracted the IcsA protein. Despite not being separated by a completed peptidoglycan wall, adjacent cells in these chains were often compartmentalized by the inner membrane, indicating that cytokinesis could occur in the absence of invagination of the entire cell envelope. Finally, deletion of penicillin-binding protein 5 from amidase mutants exacerbated the formation of twisted chains, producing numerous cells having septa with abnormal placements and geometries. The results suggest that the amidases are necessary for continued peptidoglycan synthesis during cell division, that their activities help create a septum having the appropriate geometry, and that they may contribute to the development of inert peptidoglycan.     

Mycobacterial lipid II is composed of a complex mixture of modified muramyl and peptide moieties linked to decaprenyl phosphate

Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N-acetylmuramyl-L-alanine-D-glutamate-meso-diaminopimelic acid-D-alanyl-D-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N-acetylmuramic acid, N-glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal D-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.     

N Glycolylation of the nucleotide precursors of peptidoglycan biosynthesis of Mycobacterium spp. is altered by drug treatment

The peptidoglycan of Mycobacterium spp. reportedly has some unique features, including the occurrence of N-glycolylmuramic rather than N-acetylmuramic acid. However, very little is known of the actual biosynthesis of mycobacterial peptidoglycan, including the extent and origin of N glycolylation. In the present work, we have isolated and analyzed muramic acid residues located in peptidoglycan and UDP-linked precursors of peptidoglycan from Mycobacterium tuberculosis and Mycobacterium smegmatis. The muramic acid residues isolated from the mature peptidoglycan of both species were shown to be a mixture of the N-acetyl and N-glycolyl derivatives, not solely the N-glycolylated product as generally reported. The isolated UDP-linked N-acylmuramyl-pentapeptide precursor molecules also contain a mixture of N-acetyl and N-glycolyl muramyl residues in apparent contrast to previous observations in which the precursors isolated after treatment with d-cycloserine consisted entirely of N-glycolyl muropeptides. However, nucleotide-linked peptidoglycan precursors isolated from M. tuberculosis treated with d-cycloserine contained only N-glycolylmuramyl-tripeptide precursors, whereas those from similarly treated M. smegmatis consisted of a mixture of N-glycolylated and N-acetylated residues. The full pentapeptide intermediate, isolated following vancomycin treatment of M. smegmatis, consisted of the N-glycolyl derivative only, whereas the corresponding M. tuberculosis intermediate was a mixture of both the N-glycolyl and N-acetyl products. Thus, treatment with vancomycin and d-cylcoserine not only caused an accumulation of nucleotide-linked intermediate compounds but also altered their glycolylation status, possibly by altering the normal equilibrium maintained by de novo biosynthesis and peptidoglycan recycling.     

Cell wall core galactofuran synthesis is essential for growth of mycobacteria

The mycobacterial cell wall core consists of an outer lipid (mycolic acid) layer attached to peptidoglycan via a galactofuranosyl-containing polysaccharide, arabinogalactan. This structural arrangement strongly suggests that galactofuranosyl residues are essential for the growth and viability of mycobacteria. Galactofuranosyl residues are formed in nature by a ring contraction of UDP-galactopyranose to UDP-galactofuranose catalyzed by the enzyme UDP-galactopyranose mutase (Glf). In Mycobacterium tuberculosis the glf gene overlaps, by 1 nucleotide, a gene, Rv3808c, that has been shown to encode a galactofuranosyl transferase. We demonstrate here that glf can be knocked out in Mycobacterium smegmatis by allelic replacement only in the presence of two rescue plasmids carrying functional copies of glf and Rv3808c. The glf rescue plasmid was designed with a temperature-sensitive origin of replication and the M. smegmatis glf knockout mutant is unable to grow at the higher temperature at which the glf-containing rescue plasmid is lost. In a separate experiment, the Rv3808c rescue plasmid was designed with a temperature-sensitive origin of replication and the glf-bearing plasmid was designed with a normal original of replication; this strain was also unable to grow at the nonpermissive temperature. Thus, both glf and Rv3808c are essential for growth. These findings and the fact that galactofuranosyl residues are not found in humans supports the development of UDP-galactopyranose mutase and galactofuranosyl transferase as important targets for the development of new antituberculosis drugs.     

The genetics of lantibiotic biosynthesis

The lantibiotics are a rapidly expanding group of biologically active peptides produced by a variety of Gram-positive bacteria, and are so-called because of their content of the thioether amino acids lanthionine and β-methyllanthionine. These amino acids, and indeed a number of other unusual amino acids found in the lantibiotics, arise following post-translational modification of a ribosomally synthesised precursor peptide. A number of genes involved in the biosynthesis of these highly modified peptides have been identified, including genes encoding the precursor peptide, enzymes responsible for specific amino acid modifications, proteases able to remove the leader peptide, ABC-superfamily transport proteins involved in lantibiotic translocation, regulatory proteins controlling lantibiotic biosynthesis and proteins that protect the producing strain from the action of its own lantibiotic. Analysis of these genes and their products is allowing greater understanding of the complex mechanism(s) of the biosynthesis of these unique peptides.     

Unveiling unusual features of formation of septal partition and constriction in mycobacteria--an ultrastructural study

The ultrastructural functions of the electron-dense glycopeptidolipid-containing outermost layer (OL), the arabinogalactan-mycolic acid-containing electron-transparent layer (ETL), and the electron-dense peptidoglycan layer (PGL) of the mycobacterial cell wall in septal growth and constriction are not clear. Therefore, using transmission electron microscopy, we studied the participation of the three layers in septal growth and constriction in the fast-growing saprophytic species Mycobacterium smegmatis and the slow-growing pathogenic species Mycobacterium xenopi and Mycobacterium tuberculosis in order to document the processes in a comprehensive and comparative manner and to find out whether the processes are conserved across different mycobacterial species. A complete septal partition is formed first by the fresh synthesis of the septal PGL (S-PGL) and septal ETL (S-ETL) from the envelope PGL (E-PGL) in M. smegmatis and M. xenopi. The S-ETL is not continuous with the envelope ETL (E-ETL) due to the presence of the E-PGL between them. The E-PGL disappears, and the S-ETL becomes continuous with the E-ETL, when the OL begins to grow and invaginate into the S-ETL for constriction. However, in M. tuberculosis, the S-PGL and S-ETL grow from the E-PGL and E-ETL, respectively, without a separation between the E-ETL and S-ETL by the E-PGL, in contrast to the process in M. smegmatis and M. xenopi. Subsequent growth and invagination of the OL into the S-ETL of the septal partition initiates and completes septal constriction in M. tuberculosis. A model for the conserved sequential process of mycobacterial septation, in which the formation of a complete septal partition is followed by constriction, is presented. The probable physiological significance of the process is discussed. The ultrastructural features of septation and constriction in mycobacteria are unusually different from those in the well-studied organisms Escherichia coli and Bacillus subtilis.     

Dynamics of catalysis revealed from the crystal structures of mutants of diaminopimelate epimerase

Diaminopimelate (DAP) epimerase catalyzes the stereoinversion of ll-DAP to meso-DAP, a precursor of l-lysine and an essential component of the bacterial peptidoglycan. This function is vital to bacteria and the enzyme therefore represents an attractive target for the design of novel anti-bacterials. DAP epimerase belongs to the group of PLP-independent amino acid racemases that function through a rather unusual mechanism involving two cysteines acting in concert as a base (thiolate) and an acid (thiol). We have solved the crystal structures of the apo-forms of DAP epimerase mutants (C73S and C217S) from Haemophilus influenzae at 2.3A and 2.2A resolution, respectively. These structures provide a snapshot of the enzyme in the first step of the catalytic cycle. Comparisons with the structures of the inhibitor-bound form reveal that the enzyme adopts an 'open conformation' in the absence of substrates or inhibitors with the two active site cysteines existing as a thiol-thiolate pair. Substrate binding to the C-terminal domain triggers the closure of the N-terminal domain coupled with tight encapsulation of the ligand, stabilization of the conformation of an active site loop containing Cys73 and expulsion of water molecules with concomitant desolvation of the thiolate base. This structural rearrangement is critical for catalysis.     

The alanine racemase of Mycobacterium smegmatis is essential for growth in the absence of D-alanine

Alanine racemase, encoded by the gene alr, is an important enzyme in the synthesis of d-alanine for peptidoglycan biosynthesis. Strains of Mycobacterium smegmatis with a deletion mutation of the alr gene were found to require d-alanine for growth in both rich and minimal media. This indicates that alanine racemase is the only source of d-alanine for cell wall biosynthesis in M. smegmatis and confirms alanine racemase as a viable target gene for antimycobacterial drug development.     

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.