Effect of chaotropic agents on the structure-function of recombinant acylpeptide hydrolase

Acylpeptide hydrolase, a new class the serine-type peptidase, belongs to the , hydrolase group of proteins. The tetrameric enzyme showed varying degree of stability in the presence of 1–8 M urea. The enzyme displayed about 15% of its original activity when treated with 8 M urea for 1 h at 25°C. Complete recovery of the enzyme activity was observed on dialysis or dilution (50-fold) of the denatured enzyme. However, complete abolition of the enzyme activity was observed in the presence of 1 M GnHCl. Dialysis of the 1 M GnHCl-treated enzyme resulted in 15–20% recovery of the enzyme activity. The fluorescence emission spectra of the native enzyme at 337 nm showed a red shift up to 16 nm in 8 M urea and 18 nm in the presence of 4 M GnHCl. Native enzyme during far-UV circular dichroism spectroscopy exhibited predominantly -sheet structure. The enzyme lost its secondary structure at urea concentrations of 2 M and higher, whereas the tertiary structure was minimally perturbed below 4 M urea. However, in 1 M GnHCl the enzyme lost both its secondary and tertiary structures and the enzyme was found to dissociate into monomers of 70 kDa. Both monomeric and dimeric species were observed after 24-h dialysis of the enzyme denatured with GnHCl indicating the reassociation process. Both monomer and dimers forms recovered after dialysis were active.     

Structural Investigations on Human Erythrocyte Acylpeptide Hydrolase by Mass Spectrometric Procedures

The complete primary structure of human erythrocyte acylpeptide hydrolase has been determined by using a combination of different mass spectrometric procedures and sequencing techniques. These data allowed us to correct the incomplete nucleotide sequence of the DNF15S2 locus on the short arm of human chromosome 3 at region 21, coding for the enzyme. The protein consists of 732 amino acid residues and is acetylated at the N-terminus. Alkylation experiments on the native enzyme demonstrated that all 17 cysteine residues present in the polypeptide chain are in reduced form. Multiple sequence alignment did not reveal striking similarity with proteases of known tertiary structure with the exception of members of the serine oligopeptidase family. Limited proteolysis experiments generated a C-terminal portion, containing all the catalytic triad elements responsible for proteolytic activity, and an N-terminal domain of unknown function, both still strongly associated in a completely active nicked form. The site of tryptic hydrolysis was identified as Arg193. The secondary structural organization of the protease domain of the enzyme is consistent with the / hydrolase fold.     

Expression, purification and crystal structure of a truncated acylpeptide hydrolase from Aeropyrum pernix K1

Acylpeptide hydrolase （APH） catalyzes the N-terminal hydrolysis of N^α-acylpeptides to release N^α-acylated amino acids. The crystal structure of recombinant APH from the thermophilic archaeon Aeropyrum pernix K1 （apAPH） was reported recently to be at a resolution of 2.1 A using X-ray diffraction. A truncated mutant of apAPH that lacks the first short α-helix at the N-terminal, apAPH-△（1-21）, was cloned, expressed, characterized and crystallized. Data from biochemical experiments indicate that the optimum temperature of apAPH is decreased by 15℃ with the deletion of the N-terminal α-helix. However, the enzyme activity at the optimal temperature does not change. It suggests that this N-terminal α-helix is essential for thermostability. Here, the crystal structure of apAPH-△（1-21） has been determined by molecular replacement to 2.5 A. A comparison between the two structures suggests a difference in thermostability, and it can be concluded that by adding or deleting a linking structure （located over different domains）, the stability or even the activity of an enzyme can be modified.     

Cloning, sequencing and further characterization of acylpeptide hydrolase from porcine intestinal mucosa.

Acylpeptide hydrolase was purified to homogeneity from porcine intestinal mucosa using a seven-step procedure including ammonium sulfate precipitation, gel filtration as well as anion exchange and affinity chromatography. The specific activity of the enzyme reached 105000 nmol/mg protein per min and the purification was as high as 5500-fold. This tetrameric enzyme is composed of four apparently identical subunits, the molecular mass of which was estimated to be 75 kDa, based on the results of amino acid analysis and gel electrophoresis performed under denaturing conditions. It is likely that the NH(2)-terminal residue may be acetylated, while serine was found to be the COOH-terminal residue. The hydrolytic activity of the enzyme toward N-acetyl-L-alanine p-nitroanilide at the optimum pH value was increased twofold in the presence of the chloride anion. The K(m) value calculated from the kinetics of the hydrolysis of acetylalanyl peptides was found to be 0.7+/-0.1 mM, whereas the V(max) values decreased from 200 to 50 nmol/min per microgram of enzyme, depending on the peptidic chain lengths. The V(max) value of the synthetic substrate (250 nmol/min per microgram of enzyme) was 25-500% higher than those of the acetylalanyl peptides, depending on the peptide chain length, although the enzyme affinity was slightly lower (1.8 mM as compared with 0.7 mM). In line with data on other animal species and on various tissues, the enzyme seemed likely to be a serine protease, since it was readily inhibited by diisopropyl fluorophosphate and diethyl pyrocarbonate. A 2377-nucleotide long cDNA coding for the enzyme was isolated from pig small intestine. The deduced amino acid sequence consisted of 731 residues and showed a single different amino acid with that of the porcine liver APH, except the N-terminal amino acid which is still probably lacking.     

Structural and functional insights into peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein.     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

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

Purification and properties of the allophanate hydrolase from Chlamydomonas reinhardii

Allophanate hydrolase was purified to homogeneity from extracts of Chlamydomonas reinhardii grown phototrophically using urea as sole source of nitrogen. The following sequence of steps comprised the purification procedure: (1) protamine sulfate precipitation; (2) ammonium sulfate fractionation; (3) poly(ethylene glycol) fractionation; (4) batch-wise DEAE-cellulose adsorption; (5) Sepharose 6-B gel filtration; (6) hydroxyapatite chromatography. This procedure yielded an allophanate hydrolase preparation which was homogenous as judged by polyacrylamide gel electrophoresis. The molecular weight, as determined by gradient polyacrylamide electrophoresis and gel filtration, was 110 000 and 100 000, respectively. The pH optimum of this enzyme was approximately 9.0, while the K_m for allophanate was 0.55 mM. Allophanate hydrolase was sensitive to N-ethylmaleimide but was protected from this inhibition by allophanate. Malonic acid, oxaloacetic acid, and acetoacetic acid were inhibitory to allophanate hydrolysis.     

Structural organization and analysis of the human fumarylacetoacetate hydrolase gene in tyrosinemia type I

Fumarylacetoacetate hydrolase (FAH) is a metabolic enzyme functioning at the last steo of tyrosine catabolism. Deficiency in this enzyme activity is associated with tyrosinemia type I, characterized by hypertyrosinemia, liver dysfunction, renal tubular dysfunction, liver cirrhosis, and hepatic tumors. We isolated from a human gene library a chromosomal gene related to FAH. The human FAH gene is 30 kilobases long and is split into 14 exons. All of the splice donor and acceptor sites conform to the GT/AG rule. We also analyzed findings in a patient with tyrosinemia type I with respect to the mutation responsible for detects in the enzyme. A nucleotide change from T to G was found in the exon 2 of the gene and this change was accompanied by an amino acid substitution (Phe62Cys). Transfection and expression analysis of the cDNA is cultured BMT-10 cells with the nucleotide substitution demonstrated that the substitution was indeed responsible for the decreased activity of the enzyme in the patient. These results confirmed that the T to G mutation was one of the causes of tyrosinemia type I. Structure of the FAH gene and tests for expression of the mutant FAH will facilitate further understanding of various aspects of FAH.     

Structural and Functional Studies on the Overproduced L11 Protein from Thermus thermophilus

The L11 ribosomal protein from Thermus thermophilus (TthL11) has been overproduced and purified to homogeneity using a two-step purification protocol. The overproduced protein carries a similar methylation pattern at Lys-3 as does its homolog from Escherichia coli. Chymotrypsin digested only a small part of the TthL11 protein and did not cleave TthL11 into two peptides, as in the case of EcoL11, but produced only a single N-terminal peptide. Tryptic digestion of TthL11 also produced an N-terminal peptide, in contrast to the C-terminal peptide obtained with L11 from Bacillus stearothermophilus. The recombinant protein forms a specific complex with a 55-nt 23S rRNA fragment known to interact with members of the L11 family from several organisms. Cooperative binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection of TthL11 from tryptic digestion. The similar structural and biochemical properties as well as the significant homology between L11 from E. coli and B. stearothermophilus with the corresponding protein from Thermus thermophilus indicate an evolutionarily conserved protein important for ribosome function.     

Structural properties of porcine submaxillary gland apomucin

Porcine submaxillary gland mucin was deglycosylated with a mixture of pure glycosidases to give apomucin containing less than 1% carbohydrate. The resulting apomucin freed of glycosidases was found to contain nine amino acids: threonine, serine, glutamic acid, proline, glycine, alanine, valine, isoleucine, and arginine. Serine, threonine, glycine, and alanine comprise 77% of the composition. The molecular weight of apomucin was 96,500 as determined by gel filtration in guanidine hydrochloride. Its Stokes radius was greater than 68.6 A, a far larger value than expected for a globular protein with Mr = 96,500. Circular dichroism spectroscopy of apomucin suggests that it contains 42% aperiodic or "other" structure, 40% beta-turns, 10% antiparallel pleated sheet, and 8% helical structures. The predicted secondary structure of a 50-residue peptide from ovine submaxillary gland mucin resembles the circular dichroism predictions, being dominated by turns that would lead to an extended nonglobular structure. Analysis for the secondary structure of a 36-residue tryptic peptide derived from porcine submaxillary gland apomucin predicts a similar structure. It is concluded that apomucin is likely devoid of traditional secondary structure and serves as a scaffold upon which oligosaccharides are added in O-glycosidic linkage. When sufficient sialic acid is present in the oligosaccharides, native highly viscous mucin containing about two-thirds carbohydrate by weight is obtained.     

Structural determinants of the substrate specificities of xylanases from different glycoside hydrolase families

Xylanases are of widespread importance in several food and non-food biotechnological applications. They degrade heteroxylans, a structurally heterogeneous group of plant cell wall polysaccharides, and other important components in various industrial processes. Because of the highly complex structures of heteroxylans, efficient utilization of xylanases in these processes requires an in-depth knowledge of their substrate specificity. A significant number of studies on the three-dimensional structures of xylanases from different glycoside hydrolase (GH) families in complex with the substrate provided insight into the different mechanisms and strategies by which xylanases bind and hydrolyze structurally different heteroxylans and xylo-oligosaccharides (XOS). Combined with reports on the hydrolytic activities of xylanases on decorated XOS and heteroxylans, major advances have been made in our understanding of the link between the three-dimensional structures and the substrate specificities of these enzymes. In this review, authors gave a concise overview of the structure–function relationship of xylanases from GH5, 8, 10, and 11. The structural basis for inter- and intrafamily variation in xylanase substrate specificity was discussed as are the implications for heteroxylan degradation.     

Structural studies of the Nudix hydrolase DR1025 from Deinococcus radiodurans and its ligand complexes

We have determined the crystal structure, at 1.4A, of the Nudix hydrolase DR1025 from the extremely radiation resistant bacterium Deinococcus radiodurans. The protein forms an intertwined homodimer by exchanging N-terminal segments between chains. We have identified additional conserved elements of the Nudix fold, including the metal-binding motif, a kinked beta-strand characterized by a proline two positions upstream of the Nudix consensus sequence, and participation of the N-terminal extension in the formation of the substrate-binding pocket. Crystal structures were also solved of DR1025 crystallized in the presence of magnesium and either a GTP analog or Ap(4)A (both at 1.6A resolution). In the Ap(4)A co-crystal, the electron density indicated that the product of asymmetric hydrolysis, ATP, was bound to the enzyme. The GTP analog bound structure showed that GTP was bound almost identically as ATP. Neither nucleoside triphosphate was further cleaved.     

The N-terminal nucleophile serine of cephalosporin acylase executes the second autoproteolytic cleavage and acylpeptide hydrolysis

Cephalosporin acylase (CA) precursor is translated as a single polypeptide chain and folds into a self-activating pre-protein. Activation requires two peptide bond cleavages that excise an internal spacer to form the mature αβ heterodimer. Using Q-TOF LC-MS, we located the second cleavage site between Glu(159) and Gly(160), and detected the corresponding 10-aa spacer (160)GDPPDLADQG(169) of CA mutants. The site of the second cleavage depended on Glu(159): moving Glu into the spacer or removing 5-10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of Glu. The mutant E159D was cleaved more slowly than the wild-type, as were mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a first-order, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser(170) of the β-subunit, is the nucleophile. Glu and Asp resemble glutaryl, suggesting that CA might also remove N-terminal Glu or Asp from peptides. This was indeed the case, suggesting that the N-terminal nucleophile also performed the second proteolytic cleavage. We also found that CA is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibited exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction. We propose that the final CA activation is an intra-molecular process performed by an N-terminal nucleophile, during which large conformational changes in the α-subunit C-terminal region are required to bridge the gap between Glu(159) and Ser(170).     

Preparation and characterization of des-C-terminal tubulin

Tubulin, from which the C-terminal peptide had been removed by limited proteolysis was compared to intact native tubulin. Des-C-terminal tubulin (with a nominal molecular weight of 48,000) was prepared by digestion with 1% subtilisin carlsberg at 25°C for 16 min, and the product was purified by ion-exchange chromatography on cellulose DE-52 followed by Sephadex G-50 chromatography. The purified product was composed of the cores of both the - and -subunits of tubulin and was free from other proteins and peptides containing the COOH-terminal moiety as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Sephadex G-50 and ion exchange DE-52 cellulose chromatographies, and ultracentrifugation analysis. The ultraviolet (UV) absorption and fluorescence spectra of des-C-terminal tubulin were the same as those of native tubulin. The sedimentation coefficient of des-C-terminal tubulin (5.9S) was slightly higher than that of native tubulin reflecting a decrease in axial ratio. The change in circular dichroism in the far UV indicated a decrease of -helical contents by 10–15%. These optical properties of des-C-terminal tubulin indicate that the elimination of the COOH-terminal region from tubulin did not change the conformation of the core tubulin molecule significantly, the decrease in -helix being due to the elimination of the C-terminal peptide. des-C-terminal tubulin bound 2 moles/mole of GTP and 1 mole/mole of colchicine, just as intact tubulin, but its binding ability of ruthenium red was reduced.     

Cytosolic epoxide hydrolase from liver of control and clofibrate-treated mice. Structural comparison by HPLC peptide mapping

Cytosolic epoxide hydrolases purified from livers of control and clofibrate-induced male C57B1/6 mice were compared. The proteins were reduced, alkylated and cleaved with trypsin and chymotrypsin. The digests were analyzed by HPLC and no qualitative differences were observed in the peptide mapping profiles of the two types of epoxide hydrolase preparation. The amino acid compositions and N-terminal residues of selected tryptic peptides also gave identical results for the control and clofibrate-induced mice. Both intact proteins have e-amino-blocked N-termini. The two enzyme forms are concluded to have highly similar, if not identical, primary structures.Abbreviations HPLC high-performance liquid chromatography - DABITC dimethylaminoazobenzene isothiocyanate     

Exploration of the chlorpyrifos escape pathway from acylpeptide hydrolases using steered molecular dynamics simulations

Acylpeptide hydrolases (APH) catalyze the removal of an N-acylated amino acid from blocked peptides. APH is significantly more sensitive than acetylcholinesterase, a target of Alzheimer’s disease, to inhibition by organophosphorus (OP) compounds. Thus, OP compounds can be used as a tool to probe the physiological functions of APH. Here, we report the results of a computational study of molecular dynamics simulations of APH bound to the OP compounds and an exploration of the chlorpyrifos escape pathway using steered molecular dynamics (SMD) simulations. In addition, we apply SMD simulations to identify potential escape routes of chlorpyrifos from hydrolase hydrophobic cavities in the APH-inhibitor complex. Two previously proposed APH pathways were reliably identified by CAVER 3.0, with the estimated relative importance of P1 > P2 for its size. We identify the major pathway, P2, using SMD simulations, and Arg526, Glu88, Gly86, and Asn65 are identified as important residues for the ligand leaving via P2. These results may help in the design of APH-targeting drugs with improved efficacy, as well as in understanding APH selectivity of the inhibitor binding in the prolyl oligopeptidase family.     

Structure of unsaturated rhamnogalacturonyl hydrolase complexed with substrate

Bacillus subtilis strain 168 YteR has been identified as a novel enzyme "unsaturated rhamnogalacturonyl hydrolase" classified in glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) produced from plant cell wall RG type-I treated with RG lyases, releasing unsaturated galacturonic acid (DeltaGalA) from the substrate. The most likely candidate catalytic residue is Asp-143. Here, we show the structure of D143N in complex with unsaturated RG disaccharide (substrate) determined at 1.9A resolution by X-ray crystallography. This structural feature directly contributes to the postulation of the enzyme reaction mechanism. YteR triggers the hydration of vinyl ether group in DeltaGalA, but not of glycoside bond, by using Asp-143 as a general acid and base catalyst. Asp-143 donates proton to the double bond of DeltaGalA as an acid catalyst and also deprotonates a water molecule as a base catalyst. Deprotonated water molecule attacks the C5 atom of DeltaGalA.