Characterization of proteinases from Antarctic krill (Euphausia superba)

Fractions of three trypsin-like proteinases, TL I, TL II, and TL III, a chymotrypsin-like proteinase, CL, two carboxypeptidase A enzymes, CPA I and CPA II and two carboxypeptidase B enzymes, CPB I and CPB II, from Antarctic krill (Euphausia superba) have been characterized with respect to purity by the means of capillary electrophoresis, CE, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The masses of the trypsin-like and chymotrypsin-like proteinases were determined to be 25,020, 25,070, 25,060, and 26,260Da for TL I, TL II, TL III, and CL, respectively. The masses of the CPA enzymes are likely 23,170 and 23,260Da, whereas the CPB enzyme masses likely are 33,730 and 33,900Da. The degradation efficiency and cleavage pattern of the trypsin-like proteinases were studied with native myoglobin as a model substrate using CE, MALDI-TOF-MS, and nanoelectrospray mass spectrometry (nESI-MS). The degradation efficiency of the trypsin-like proteinases was found to be approximately 12 and 60 times higher compared to bovine trypsin at 37 degrees C and 1-3 degrees C, respectively. All three fractions of trypsin-like proteinases showed a carboxypeptidase activity in combination with their trypsin activity.     

Label-Free Quantitative Proteomics Unravels Carboxypeptidases as the Novel Biomarker in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with a high mortality rate and poor prognosis. However, little is known concerning the molecular mechanism of PDAC at the proteomics level. Here we report a proteomics analysis of PDAC tumor and adjacent tissues by shotgun proteomics followed by label-free quantification, and in total, 3031 and 3306 proteins were identified in three pairs of PDAC tumor and adjacent tissues, respectively; 40 of them were differentially expressed for at least three-fold in PDAC tumor tissues. Ontological and interaction network analysis highlighted the dysregulation of a set of four proteins in the carboxypeptidase family: carboxypeptidase A1 (CPA1), A2 (CPA2), B1 (CPB1), and chymotrypsin C (CTRC). Western blotting confirmed the downregulation of the carboxypeptidase network in PDAC. Immunohistochemistry of tissue microarray from 90 PDAC patients demonstrated that CPB1 was downregulated 7.07-fold (P < .0001, n = 81) in tumor comparing with the peritumor tissue. Further 208 pancreatic tissues from PDAC tumor, peritumor, and pancreatis confirmed the downregulation of CPB1 in the PDAC patients. In summary, our results displayed that the expression of carboxypeptidase is significantly downregulated in PDAC tumor tissues and may be novel biomarker in the patient with PDAC.     

Stabilisation of carboxypeptidases in acid extracts of ovine pancreas

Carboxypeptidases A (CPA) and B (CPB) are pancreatic exopeptidases that are very sensitive to environmental stresses such as freezing, drying, temperature and pH. Traditional low pH methods of extracting pancreatic enzymes destroy carboxypeptidase activity. The use of sucrose as a stabilising agent prevents pH induced denaturation of carboxypeptidases during their extraction. Greater than 70% carboxypeptidase A activity can be retained at pHs as low as 3.5 using 0.5 M sucrose. In addition extraction at low pH ensures good trypsin and chymotrypsin recovery while elastase recovery is improved due to the stabilising effect of sucrose. Further fractionation of the extract produces preparations enriched in endopeptidases or exopeptidase suitable for use in protein hydrolysis applications.     

Digestive proteolysis in the Colorado potato beetle,Leptinotarsa decemlineata: Activity-based profiling and imaging of a multipeptidase network

The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major pest of potato plants, and its digestive system is a promising target for development of pest control strategies. This work focuses on functional proteomic analysis of the digestive proteolytic enzymes expressed in the CPB gut. We identified a set of peptidases using imaging with specific activity-based probes and activity profiling with selective substrates and inhibitors. The secreted luminal peptidases were classified as: (i) endopeptidases of cathepsin D, cathepsin L, and trypsin types and (ii) exopeptidases with aminopeptidase (cathepsin H), carboxypeptidase (serine carboxypeptidase, prolyl carboxypeptidase), and carboxydipeptidase (cathepsin B) activities. The proteolytic arsenal also includes non-luminal peptidases with prolyl oligopeptidase and metalloaminopeptidase activities. Our results indicate that the CPB gut employs a multienzyme network of peptidases with complementary specificities to efficiently degrade ingested proteins. This proteolytic system functions in both CPB larvae and adults and is controlled mainly by cysteine and aspartic peptidases and supported by serine and metallopeptidases. The component enzymes identified here are potential targets for inhibitors with tailored specificities that could be engineered into potato plants to confer resistance to CPB.     

Localization of a highly immunogenic region of carboxypeptidase A recognized by three different monoclonal antibodies and their use in the detection of subtle conformational alterations in this enzyme region

Three murine monoclonal antibodies (mAb 100, 104, and 121) elicited against carboxypeptidase A (CPA) were prepared and characterized. All three mAbs recognize the same or partially overlapping sites of CPA. This is corroborated by the lack of antibody additivity in the ELISA assay carried out in the presence of pairs of mAbs, the similarity in molecular weight of the immunocomplex formed between CPA and one of the mAbs in the presence of another, and also a competition experiment in which one of the mAbs was labeled enzymatically. The three mAbs do not affect the enzymatic activity of CPA. Even at high concentrations, they do not recognize carboxypeptidase B (CPB) in spite of the similar tertiary structure and the 50% homology in amino acid sequence with CPA. This antigenic determinant is located on one of the four cyanogen bromide fragments of CPA. On the basis of the known sequences of the two enzymes, criteria which predict high antigenicity, and experimental data using synthetic peptides, such a determinant was found to be located within the amino acid sequence from residues 209 to 218 of the CPA molecule. The mAbs prepared detect conformational alterations in the above enzyme epitope when the enzyme is exposed to various conditions. The binding of the mAbs to CPA adsorbed onto a polystyrene plate is characterized by apparent binding constants higher by 1 or 2 orders of magnitude than those characterizing the interaction of the mAbs with CPA in solution. The mAbs also readily detect both conformational alterations of CPA on treatment with urea and subtle, reversible conformational alterations on removal of zinc from the active site of the enzyme.     

Molecular cloning and cDNA sequence analysis of carboxypeptidases A1, A2 and B from the Japanese flounder Paralichthys olivaceus

Although pancreatic serine proteases have been cloned in teleosts, no sequence data are currently available on members of the carboxypeptidase (CP) family. Here, we cloned cDNAs coding for two preproCPAs, corresponding to mammalian preproCPA1 and preproCPA2, and one preproCPB from a pancreatic cDNA library of the Japanese flounder, Paralichthys olivaceus. The activation peptides of flounder proCPs completely retained the sequences for inhibition of enzymatic activity of proCPs just like mammalian proCPs. Of 306–309 amino acids in total, 95 amino acids are completely conserved between bovine CPA1 and CPB and flounder CPs. Notably, amino acid residues for Zn²⁺ ligands, catalysis and substrate anchoring are completely conserved between flounder and bovine CPs. Three species of flounder preproCPs are all expressed in the pancreas of first feeding larvae.     

A novel rat carboxypeptidase, CPA2: characterization, molecular cloning, and evolutionary implications on substrate specificity in the carboxypeptidase gene family

A new member of the carboxypeptidase gene family, carboxypeptidase A2 (CPA2), has been identified from the predicted amino acid sequence of a rat pancreatic cDNA clone. In vivo recombination and in situ hybridization techniques employing the CPA2 cDNA resulted in the isolation of two genomic clones spanning the 25-kilobase pair rat CPA2 gene. Evolutionary trees built from the amino acid sequences of the known pancreatic carboxypeptidases show that CPA2 and carboxypeptidase A1 (CPA1) are the products of genes which duplicated before the mammalian radiation, and that bovine CPA is of the A1 type. The substrate specificities of CPA1 and CPA2 isolated from rat pancreas are similar to bovine CPA in that carboxyl-terminal amino acids with aromatic or branched aliphatic side chains are preferred. However, the substrate preference of rat CPA1 is skewed toward smaller amino acids, while that of rat CPA2 is skewed toward bulkier amino acids as compared to bovine CPA. The differences in the substrate specificities of these three carboxypeptidases are compatible with the nature of the amino acid replacements in their binding pockets for the carboxylterminal amino acid of the substrate.     

Carboxypeptidase O is a glycosylphosphatidylinositol-anchored intestinal peptidase with acidic amino acid specificity

The first metallocarboxypeptidase (CP) was identified in pancreatic extracts more than 80 years ago and named carboxypeptidase A (CPA; now known as CPA1). Since that time, seven additional mammalian members of the CPA subfamily have been described, all of which are initially produced as proenzymes, are activated by endoproteases, and remove either C-terminal hydrophobic or basic amino acids from peptides. Here we describe the enzymatic and structural properties of carboxypeptidase O (CPO), a previously uncharacterized and unique member of the CPA subfamily. Whereas all other members of the CPA subfamily contain an N-terminal prodomain necessary for folding, bioinformatics and expression of both human and zebrafish CPO orthologs revealed that CPO does not require a prodomain. CPO was purified by affinity chromatography, and the purified enzyme was able to cleave proteins and synthetic peptides with greatest activity toward acidic C-terminal amino acids unlike other CPA-like enzymes. CPO displayed a neutral pH optimum and was inhibited by common metallocarboxypeptidase inhibitors as well as citrate. CPO was modified by attachment of a glycosylphosphatidylinositol membrane anchor to the C terminus of the protein. Immunocytochemistry of Madin-Darby canine kidney cells stably expressing CPO showed localization to vesicular membranes in subconfluent cells and to the plasma membrane in differentiated cells. CPO is highly expressed in intestinal epithelial cells in both zebrafish and human. These results suggest that CPO cleaves acidic amino acids from dietary proteins and peptides, thus complementing the actions of well known digestive carboxypeptidases CPA and CPB.     

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis)

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.     

Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

Thermostabilization of carboxypeptidase A by interaction with its monoclonal antibodies.

The effect of interaction of carboxypeptidase A (CPA) with three monoclonal antibodies, each with a different epitope (CP 10, CP 9, and CP 8), on the heat stabilization of enzymes is described. These monoclonal antibodies bind to CPA with a relatively high binding constant (approximately 10(8) M-1) and do not affect its catalytic properties. Intact carboxypeptidase A lost more than 95 and 90% of its esterase and peptidase activities within 120 min at 50 degrees C. The monoclonal antibodies increased the thermal stability of the enzyme by 90 and 60%, as compared with the peptidase and esterase initial activities, respectively. Binding of these monoclonal antibodies, alone or in pairs, to the enzyme epitopes that are supposedly involved in heat denaturation of CPA result in stabilization of the conformation of the enzyme. The effect of thermostabilization by monoclonal antibodies was more pronounced with respect to peptidase activity than to esterase activity, indicating that these activities follow different reaction mechanisms. Since properly selected monoclonal antibodies can be prepared against virtually any enzyme, their immunocomplexation may provide a general and convenient method for stabilization of the enzyme conformation to heat denaturation, without affecting the catalytic properties.     

Early postnatal development of peptide hydrolysis in chicks and guinea pigs.

1. To characterize the development of peptide hydrolysis the activities of pancreatic carboxypeptidase A (CPA) and intestinal glycylleucine dipeptidase (GLDP) were registered in 1-45 days old chicks, as well as GLDP activities in newborn and adult guinea pigs. 2. The highest values of CPA and GLDP relative activities were found immediately after hatching, maximal decrease of activities took place during the first week. 3. GLDP activities gradient on the surface of the small intestine of chicks has two maximums: one in the upper jejunum, the other one--in the lower ileum. The development of proximo-distal gradient began at the age of 7 days and finished at the end of the first month. 4. Total CPA and GLDP activities decreased during the first week; up to the 15-20 day they reached the initial level and later exceeded it. 5. Relative GLDP activity in guinea pigs declined with aging, while the total activity increased, as it was demonstrated for chicks. 6. GLDP activity was distributed equally along the surface of the small intestine in newborn guinea pigs as well as in mature animals.     

Structural characterization of the rat carboxypeptidase A1 and B genes. Comparative analysis of the rat carboxypeptidase gene family

Nucleotide sequencing of a rat carboxypeptidase B (CPB) cDNA and direct sequencing of the CPB mRNA via primer extension on pancreatic polyadenylated RNA has yielded the complete amino acid sequence of rat CPB. The rat enzyme is synthesized as a precursor species containing a large amino-terminal fragment (108 amino acids) that contributes a putative signal sequence and an activation peptide. The mature form of rat CPB is homologous to bovine CPB (77% identity); the amino acids in bovine CPB which have been previously implicated in catalysis or ligand binding are invariant in the rat orthologue. The rat CPB cDNA was used as a probe for the isolation of the rat CPB gene. Detailed characterization of three overlapping rat genomic clones demonstrated that the coding region for the rat CPB precursor is sequestered in 11 exons which are dispersed throughout 34 kilobase pairs of genomic DNA. The nucleotide sequence of a large part of the gene has been determined including that of the exons, the exon/intron boundaries, and the 5' flanking region. We also report the partial nucleotide sequence of the rat CPA1 gene. Comparative analysis of the structural organization of the rat CPB, rat CPA1, and rat CPA2 genes (Gardell, S. J., Craik, C. S., Clauser, E., Goldsmith, E. J., Stewart, C.-B., Graf, M., and Rutter, W. J. (1988) J. Biol. Chem. 263, 17828-17836) reveals that, with one exception, the number, position, and sequence composition of the exons in these three carboxypeptidase genes are conserved in spite of considerable divergence with respect to the lengths of their corresponding intervening sequences. Conserved sequences in the 5' flanking regions of the rat CPA1, CPA2, CPB, and other pancreas-specific genes have been identified.     

Tubulinyl-tyrosine Carboxypeptidase from Chicken Brain: Properties and Partial Purification

Abstract: Tyrosine can be released from tubulinyl-tyrosine by the action of a brain carboxypeptidase. The molecular weight of this enzyme found by gel filtration through a column of Sephadex G-200 was 90,000. The enzyme was very unstable in a purified preparation in which the activity per milligram of protein was increased 250-fold with respect to the starting material. The precise magnitude of the purification cannot be stated because of the unknown amount of endogenous tubulinyl-tyrosine in the material to be assayed. A comparative study was done between tubulinyl-tyrosine carboxypeptidase (TTCP) activity and pancreatic carboxypeptidase A (CPA, EC 3.4.12.2) activity using tubulinyl-[¹⁴C]tyrosine as substrate. The most remarkable differences found are: MgCl₂ (2 mM), phenyl acetate (10 mM), or EDTA (5 mM) increased the TTCP activity whereas the CPA activity was strongly inhibited by these compounds, lodoacetate (2 mM) and ZnCl₂ (0.1 mM) inhibited the TTCP activity more than the CPA activity. Contrarily, mercaptoethanol (50 mM) and dimethyl sulfoxide (5%) showed a stronger inhibitory effect on CPA than on TTCP. Of several N-carbobenzoxy dipeptides (Z-dipeptides) tested the greatest inhibitory effects on TTCP activity were obtained with Z-Glu-Tyr and Z-Glu-Phe, although strong inhibitory effects on CPA were also obtained with other Z-dipeptides.     

The intrinsic threshold of the fibrinolytic system is modulated by basic carboxypeptidases, but the magnitude of the antifibrinolytic effect of activated thrombin-activable fibrinolysis inhibitor is masked by its instability

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is intrinsically unstable, a property that complicates the study of its role in regulating fibrinolysis. To investigate the effect of basic carboxypeptidases on fibrinolysis under conditions of constant carboxypeptidase activity, we employed pancreatic carboxypeptidase B (CPB), a homologous, stable basic carboxypeptidase, as a surrogate for TAFIa. Clots formed from TAFI-depleted plasma or from purified components were supplemented with tissue-type plasminogen activator and either CPB or TAFIa. The clot lysis data indicate that the down-regulation of fibrinolysis mediated by basic carboxypeptidases involves a threshold mechanism. At carboxypeptidase concentrations above the threshold, plasminogen activation is maintained in a fully down-regulated state; experiments in plasma showed that fibrinolysis is essentially halted by saturating concentrations of TAFIa and that fibrinolysis can be prolonged more than 45-fold by a stable carboxypeptidase. The threshold carboxypeptidase concentration was dependent on tissue-type plasminogen activator and antiplasmin concentrations, indicating that the threshold is determined by the steady-state plasmin concentration. Although obvious with CPB, the threshold was masked by the intrinsic instability of TAFIa and became apparent only when the effect of TAFIa was investigated over the picomolar concentration range. Because of the threshold effect and the instability of TAFIa, exponential increases in TAFIa concentration generate linear increases in lysis time. A model relating lysis time to TAFIa concentration, TAFIa half-life, and the threshold concentration of TAFIa is provided. The threshold effect has potentially important implications regarding the role of TAFIa and the regulation of clot lysis in vivo.     

Thioxophosphoranyl aryl- and heteroaryloxiranes as the representants of a new class of metallocarboxypeptidase inhibitors

A novel and potent family of metallocarboxypeptidase inhibitors based on thioxophosphoranyl oxiranes is presented. These compounds bear aryl or heteroaryl substituents with trans-stereochemistry with respect to the phosphorylated group and they have been synthesized by the addition of [bis(diisopropylamino)phosphino](trimethylsilyl)carbene to the corresponding aldehydes and the subsequent thiolation of the phosphine. These oxiranes contain a tetrahedral P atom harboring shielded N,N-groups. The screening of their biological activity as metallocarboxypeptidase inhibitors and some structural studies, as well as full experimental details for the new compounds, is disclosed. Thus, from the analysis of their activity against the prototypical metallocarboxypeptidases A and B (CPA and CPB), we have observed that hydrophobic phosphorylated oxiranes perform better as CPB inhibitors, reaching K_i values comparable to classical synthetic carboxypeptidase inhibitors. X-ray diffraction analysis revealed that the packing in the structure of one phosphorylated oxirane is mediated mainly by hydrophobic contacts and that the N,N-groups are highly flexible. Consequently, phosphorylated oxiranes might constitute an attractive material for subsequent improvements in the design of novel inhibitors against human proteolytic enzymes with enhanced oral availability.     

Preliminary characterization of enzyme activities in Malpighian tubules involved in the breakdown of adipokinetic hormones

Adipokinetic hormones (AKH) from different insect species, crustacean red pigment-concentrating hormone (RPCH), and synthetic substrates were used to characterized enzyme activities present in the Malpighian tubules (MT) of the desert locuts, Schistocerca gregaria, which are involved in the degradation of AKH. When peptides containing proline (position 6) were incubated with MT homogenate they were cleaved by a post-proline cleaving enzyme (PPCE). The presence of such an enzyme was confirmed by the breakdown of a synthetic substrate for PPCE. Peptides which do not contain proline were broken down by a post-phenylalanine cleaving enzyme (PFCE) which could be chymotrypsin or chymotryptic. This PFCE activity(ies) seem(s) to be inactive on the proline-containig peptides or their fragments or digests these at a slow rate. The C-terminal chymotrypsin fragments of the AKHs were broken down by MT homogenates with no accumulation of new intermediate products. It is not clear whether another endopeptidase, PPCE, or leucine aminopeptidase (LAP) is responsible. The MTs contain LAP activity; however, this enzyme(s) may be different from its vertebrate counterpart(s). Homogenates of MTs break down equimolar amounts of Pro-7AMC at approximately the same rate, while porcine kidney LAP (cytosol) cleaved Pro-7AMC much slower than Leu-7AMC. The demonstration of carboxypeptidase (CP) A and B activity in the MTs was not possible using conventional substrates such as hippuryl derivatives of amino acids. When CPA from porcine pancreas was added to MT homogenates hippuryl-phenylalanine was digested proving that the conditions were appropriate for CPA activity to occur. The treatment of a N-terminally blocked peptide fragment with MT homogenate led to the breakdown of the peptide giving evidence that the MT CP requires a substrate with a somewhat longer length of amino acid residues.     

Belactins A and B,New Serine Carboxypeptidase Inhibitors Produced by Actinomycete. I. Taxonomy,Production, Isolation and Biological Actmties

Abstract

Belactins A and B, new inhibitors of serine carboxypeptidase were discovered in the fermentation broth of Saccharopolyspora sp. MK19–42F6. They were purified by ethyl acetate extraction, silica gel chromatography, Sephadex LH20 chromatography, Capcellpak C18 SG120 reversed phase HPLC and centrifugal partition chromatography (CPC) following their inhibitory activity against carboxypeptidase Y (CP-Y). The inhibition constants (K_i) of belactins A and B against CP-Y are 0.14 and 0.27 μM respectively. Belactins A and B have highly specific inhibitory activities for CP-Y among various peptidases, have no antimicrobial activities at 100 μ/ml and have low toxicities.     

The role of the S1 binding site of carboxypeptidase M in substrate specificity and turn-over

The influence of the P1 amino acid on the substrate selectivity, the catalytic parameters K(m) and k(cat), of carboxypeptidase M (CPM) (E.C. 3.4.17.12) was systematically studied using a series of benzoyl-Xaa-Arg substrates. CPM had the highest catalytic efficiency (k(cat)/K(m)) for substrates with Met, Ala and aromatic amino acids in the penultimate position and the lowest with amino acids with branched side-chains. Substrates with Pro in P1 were not cleaved in similar conditions. The P1 substrate preference of CPM differed from that of two other members of the carboxypeptidase family, CPN (CPN/CPE subfamily) and CPB (CPA/CPB subfamily). Aromatic P1 residues discriminated most between CPM and CPN. The type of P2 residue also influenced the k(cat) and K(m) of CPM. Extending the substrate up to P7 had little effect on the catalytic parameters. The substrates were modelled in the active site of CPM. The results indicate that P1-S1 interactions play a role in substrate binding and turn-over.     

First hydroxamate inhibitors for carboxypeptidase A. N-acyl-N-hydroxy-beta-phenylalanines

A series of N-acyl-N-hydroxy-beta-Phe were designed, synthesized, and shown to have potent inhibitory activity for carboxypeptidase A (CPA). They are the first examples of CPA inhibitors having a hydroxamate functionality.