Crystal structure of NAD+-dependent Peptoniphilus asaccharolyticus glutamate dehydrogenase reveals determinants of cofactor specificity

Glutamate dehydrogenases (EC 1.4.1.2-4) catalyse the oxidative deamination of l-glutamate to α-ketoglutarate using NAD(P) as a cofactor. The bacterial enzymes are hexamers and each polypeptide consists of an N-terminal substrate-binding (Domain I) followed by a C-terminal cofactor-binding segment (Domain II). The reaction takes place at the junction of the two domains, which move as rigid bodies and are presumed to narrow the cleft during catalysis. Distinct signature sequences in the nucleotide-binding domain have been linked to NAD(+) vs. NADP(+) specificity, but they are not unambiguous predictors of cofactor preferences. Here, we have determined the crystal structure of NAD(+)-specific Peptoniphilus asaccharolyticus glutamate dehydrogenase in the apo state. The poor quality of native crystals was resolved by derivatization with selenomethionine, and the structure was solved by single-wavelength anomalous diffraction methods. The structure reveals an open catalytic cleft in the absence of substrate and cofactor. Modeling of NAD(+) in Domain II suggests that a hydrophobic pocket and polar residues contribute to nucleotide specificity. Mutagenesis and isothermal titration calorimetry studies of a critical glutamate at the P7 position of the core fingerprint confirms its role in NAD(+) binding. Finally, the cofactor binding site is compared with bacterial and mammalian enzymes to understand how the amino acid sequences and three-dimensional structures may distinguish between NAD(+) vs. NADP(+) recognition.     

Inherent chaperone-like activity of aspartic proteases reveals a distant evolutionary relation to double-psi barrel domains of AAA-ATPases

Chaperones and proteases share the ability to interact with unfolded proteins. Here we show that enzymatically inactive forms of the aspartic proteases HIV-1 protease and pepsin have inherent chaperone-like activity and can prevent the aggregation of denatured substrate proteins. In contrast to proteolysis, which requires dimeric enzymes, chaperone-like activity could be observed also with monomeric domains. The involvement of the active site cleft in the chaperone-like function was demonstrated by the inhibitory effect of peptide substrate inhibitors. The high structural similarity between aspartic proteases and the N-terminal double-psi barrels of Cdc48-like proteins, which are involved in the unfolding and dissociation of proteins, suggests that they share a common ancestor. The latent chaperone-like activity in aspartic proteases can be seen as a relic that has further evolved to serve substrate binding in the context of proteolytic activity.     

Isolation, purification, and characterization of phenylpyruvate transaminating enzymes of Erwinia carotovora

Enzymes of Erwinia carotovora that transaminate phenylpyruvate were isolated, purified, and characterized. Two aromatic aminotransferases (PAT1 and PAT2) and an aspartic aminotransferase (PAT3) were found. According to gel filtration, these enzymes have molecular weights of 76, 75, and 78 kDa. The enzymes consist of two identical subunits of molecular weights of 31.4, 31, and 36.5 kDa, respectively. The isoelectric points of PAT1, PAT2, and PAT3 were determined as 3.6, 3.9, and 4.7, respectively. The enzyme preparations considerably differ in substrate specificity. All three of the enzymes productively interacted with the following amino acids: L-aspartic acid, L-leucine (except PAT3), L-isoleucine (except PAT3), L-serine, L-methionine, L-cysteine, L-phenylalanine, L-tyrosine, and L-tryptophane. The aromatic aminotransferases display higher specificity to the aromatic amino acids and the leucine-isoleucine pair, whereas the aspartic aminotransferase displays higher specificity to L-aspartic acid and relatively low specificity to the aromatic amino acids. The aspartic aminotransferase does not use L-leucine or L-isoleucine as a substrate. PAT1, PAT2, and PAT3 show the highest activity at pH 8.9 and at 48, 53, and 58°C, respectively.     

Mapping selectivity and specificity of active site of plasmepsins from Plasmodium falciparum using molecular interaction field approach

In the search for selectivity, the aspartic proteases are known to be a very difficult case because the enzymes of this family are not only sequentially but structurally also very similar. To gain insight into the selectivity and specificity of the aspartic proteases family we characterized the binding sites of four malarial aspartic protease (plasmepsin I, plasmepsin II, plasmepsin IV, P. vivax plasmepsin) and two human aspartic proteases (cathepsin D and pepsin) with the intention of identifying the regions that could be potential sites for obtaining selectivity using molecular interaction field approach.     

Structure of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: implications for the design of antifungal agents.

The three-dimensional structure of a secreted aspartic protease from Candida albicans complexed with a potent inhibitor reveals variations on the classical aspartic protease theme that dramatically alter the specificity of this class of enzymes. The structure presents: (1) an 8-residue insertion near the first disulfide (Cys 45-Cys 50, pepsin numbering) that results in a broad flap extending toward the active site; (2) a 7-residue deletion replacing helix hN2 (Ser 110-Tyr 114), which enlarges the S3 pocket; (3) a short polar connection between the two rigid body domains that alters their relative orientation and provides certain specificity; and (4) an ordered 11-residue addition at the carboxy terminus. The inhibitor binds in an extended conformation and presents a branched structure at the P3 position. The implications of these findings for the design of potent antifungal agents are discussed.     

Key features determining the specificity of aspartic proteinase inhibition by the helix-forming IA3 polypeptide

The 68-residue IA(3) polypeptide from Saccharomyces cerevisiae is essentially unstructured. It inhibits its target aspartic proteinase through an unprecedented mechanism whereby residues 2-32 of the polypeptide adopt an amphipathic alpha-helical conformation upon contact with the active site of the enzyme. This potent inhibitor (K(i) < 0.1 nm) appears to be specific for a single target proteinase, saccharopepsin. Mutagenesis of IA(3) from S. cerevisiae and its ortholog from Saccharomyces castellii was coupled with quantitation of the interaction for each mutant polypeptide with saccharopepsin and closely related aspartic proteinases from Pichia pastoris and Aspergillus fumigatus. This identified the charged K18/D22 residues on the otherwise hydrophobic face of the amphipathic helix as key selectivity-determining residues within the inhibitor and implicated certain residues within saccharopepsin as being potentially crucial. Mutation of these amino acids established Ala-213 as the dominant specificity-governing feature in the proteinase. The side chain of Ala-213 in conjunction with valine 26 of the inhibitor marshals Tyr-189 of the enzyme precisely into a position in which its side-chain hydroxyl is interconnected via a series of water-mediated contacts to the key K18/D22 residues of the inhibitor. This extensive hydrogen bond network also connects K18/D22 directly to the catalytic Asp-32 and Tyr-75 residues of the enzyme, thus deadlocking the inhibitor in position. In most other aspartic proteinases, the amino acid at position 213 is a larger hydrophobic residue that prohibits this precise juxtaposition of residues and eliminates these enzymes as targets of IA(3). The exquisite specificity exhibited by this inhibitor in its interaction with its cognate folding partner proteinase can thus be readily explained.     

Activation of methionine by Escherichia coli methionyl-tRNA synthetase

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of a precursor to the aspartic proteinase from Rhizomucor miehei shows that the enzyme is synthesized as a zymogen

In order to characterize the zymogen of the milk-clotting enzyme from Rhizomucor miehei, we constructed a cDNA library on pBR327 in Escherichia coli. Aspartic proteinase-specific recombinants were isolated by colony hybridization to a specific oligonucleotide mixture, and the cDNA sequence corresponding to a precursor form of the enzyme was determined. The deduced amino acid sequence shows that this secreted fungal proteinase is synthesized as a precursor. The first 22 amino acid residues in this precursor constitute a typical signal peptide. The amino acid sequence of the following 47-amino-acid-long prosegment shows homology to the prosegments from both the extracellular and intracellular vertebrate aspartic proteinases, and to the prosegments from the yeast and Mucor pusillus aspartic proteinases as well. These observations suggest that all aspartic proteinases are synthesized with a prosegment and that this prosegment is essential for the correct folding of all the mature enzymes. The active Rhizomucor miehei enzyme consists of 361 amino acid residues with a total molecular weight of 38,701. Clusters of identities around the active site cleft support the assumption that these proteinases have a common folding of their peptide chains. The disulphide bridges were localized in the fungal enzyme, and 2 N-glycosylation sites were identified.     

The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis.

Subtilisin from a wide variety of Bacillus species has been extensively investigated as a promising target for protein engineering. In this study, we analyzed the substrate specificity of B. subtilis subtilisin E based on the structure of a new alkaline elastase produced by the alkalophilic Bacillus strain Ya-B, which has very high elastolytic activity. Despite the high homology of the primary sequences of both enzymes (54% identical), alkaline elastase was found to lack four consecutive amino acids which, in subtilisin, have been shown by X-ray analysis to lie close to the P1 binding cleft. To examine the influence of such a deletion in subtilisin on its substrate specificity, we constructed several mutants missing four amino acids by site-directed mutagenesis. When assayed with synthetic peptides, elastin and casein as substrates, a mutant lacking Ser161-Thr162-Ser163-Thr164 showed considerably lower specific activity toward the substrates for subtilisin, and its substrate specificity approached that of alkaline elastase. The results indicate that the deletion in subtilisin E influences the catalytic efficiency as well as the P1 specificity, and that this region is, in part, responsible for the difference in specificity between the two enzymes.     

Comparative molecular modeling analysis of­5‐amidinoindole and benzamidine binding to thrombin and trypsin: specific H‐bond formation contributes to high 5‐amidinoindole potency and selectivity for thrombin and factor Xa

The coagulation cascade enzymes thrombin and factor Xa are known to have specificity pockets very similar to those of trypsin and plasmin. However, comparative molecular modeling analysis of the crystal structures of benzamidine–thrombin and benzamidine–trypsin, in conjunction with a docking analysis of 5‐amidinoindole and related inhibitors in both enzymes reveals subtle differences between the specificity sites of the two types of enzymes. Specifically, thrombin and factor Xa, which have an alanine residue at position 190, exhibit increased activities for the rigid and more bulky bicyclic derivatives of benzamidine (e.g. amidinobenzofuran, amidinothiophene and amidinoindole), because of additional hydrophobic and H‐bond interactions between the inhibitors and the specificity sites, whereas enzymes with a serine residue at position 190, like trypsin and plasmin, exhibit little difference in activity among the same set of compounds because of the orientational restriction imposed on the inhibitors by Ser190, which forms an additional H‐bond with the amidino group of the inhibitors. Enzymes of both groups show similar responses to the flexible aminobenzamidine since the smaller size and the rotatable anilino group of the inhibitor would allow the inhibitor to achieve favorable electrostatic interactions with both groups of enzymes. 5‐Amidinoindole is the most dramatic example of the rigid bicyclic type inhibitor. Based on our docking analysis, we propose that a selective H‐bond with the hydroxyl group of the catalytic Ser195 and the subtle differences in steric fit imposed by Ala/Ser at position 190 explain the high potency and selectivity of 5‐amidinoindole for thrombin and factor Xa. Copyright © 1999 John Wiley & Sons, Ltd.     

Biosynthesis of lysosomal endopeptidases

Despite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse-chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino-terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high-mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino-terminal activation peptides. Removal of the propeptide generates an active single-chain enzyme. Whether the single-chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to the lysosome.     

Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes

The hydrolases and transferases that constitute the alpha-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (beta/alpha)(8)-barrel, with the active site being at the C-terminal end of the barrel beta-strands. Although the enzymes are believed to share the same catalytic acids and a common mechanism of action, they have been assigned to three separate families - 13, 70 and 77 - in the classification scheme for glycoside hydrolases and transferases that is based on amino acid sequence similarities. Each enzyme has one glutamic acid and two aspartic acid residues necessary for activity, while most enzymes of the family also contain two histidine residues critical for transition state stabilisation. These five residues occur in four short sequences conserved throughout the family, and within such sequences some key amino acid residues are related to enzyme specificity. A table is given showing motifs distinctive for each specificity as extracted from 316 sequences, which should aid in identifying the enzyme from primary structure information. Where appropriate, existing problems with identification of some enzymes of the family are pointed out. For enzymes of known three-dimensional structure, action is discussed in terms of molecular architecture. The sequence-specificity and structure-specificity relationships described may provide useful pointers for rational protein engineering.     

Screening combinatorial libraries by mass spectrometry. 2. Identification of optimal substrates of protein tyrosine phosphatase SHP-1

Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. In this work, we have developed a novel combinatorial library method, termed "enzyme-catalyzed loss of isotope peak signal enhancement (ECLIPSE)", to determine the substrate specificity of PTPs. This method involves partial labeling of pY at a nonbridging phosphate oxygen atom with 50% (18)O ((16)O/(18)O = 1:1). A 361-member solution-phase peptide library with randomization at the -1 and -2 positions (relative to pY), RNNXXpYA-NH(2) (X = 19 alpha-amino acids except for Cys), was synthesized with the partially (18)O-labeled pY by the split-synthesis method. Each member of the resulting pY peptide library appeared as a doublet peak in the mass spectrum (m/z m and m + 2.0043). Limited treatment of the library with a PTP removed the mass-degenerate phosphoryl group from the most preferred substrates to generate products as singlet peaks, which were readily identified and sequenced by tandem mass spectrometry. Screening of the pY library against the catalytic domain of SHP-1 revealed that SHP-1 prefers an acidic residue at the -2 position, with aspartic acid being slightly better than glutamic acid. At the -1 position, SHP-1 also prefers an acidic residue, although a variety of other amino acids are also tolerated. On the other hand, positively charged residues at these positions render the corresponding peptides very poor substrates of SHP-1. Several selected peptides were individually synthesized and assayed against SHP-1, and the kinetic data confirmed the screening results. These results demonstrate that ECLIPSE is a viable method for studying the substrate specificity of PTPs.     

Crystal structure of cod liver class I alcohol dehydrogenase: substrate pocket and structurally variable segments.

The structural framework of cod liver alcohol dehydrogenase is similar to that of horse and human alcohol dehydrogenases. In contrast, the substrate pocket differs significantly, and main differences are located in three loops. Nevertheless, the substrate pocket is hydrophobic like that of the mammalian class I enzymes and has a similar topography in spite of many main-chain and side-chain differences. The structural framework of alcohol dehydrogenase is also present in a number of related enzymes like glucose dehydrogenase and quinone oxidoreductase. These enzymes have completely different substrate specificity, but also for these enzymes, the corresponding loops of the substrate pocket have significantly different structures. The domains of the two subunits in the crystals of the cod enzyme further differ by a rotation of the catalytic domains by about 6 degrees. In one subunit, they close around the coenzyme similarly as in coenzyme complexes of the horse enzyme, but form a more open cleft in the other subunit, similar to the situation in coenzyme-free structures of the horse enzyme. The proton relay system differs from the mammalian class I alcohol dehydrogenases. His 51, which has been implicated in mammalian enzymes to be important for proton transfer from the buried active site to the surface is not present in the cod enzyme. A tyrosine in the corresponding position is turned into the substrate pocket and a water molecule occupies the same position in space as the His side chain, forming a shorter proton relay system.     

Interconversion between serine and aspartic acid in the alpha helix of the N-terminal zinc finger of Sp1: implication for general recognition code and for design of novel zinc finger peptide recognizing complementary strand

In the typical base recognition mode of the C(2)H(2)-type zinc finger, the amino acid residues at alpha-helical positions -1, 3, and 6 make a contact with the base in one strand (the primary strand), and the residue at position 2 interacts with the base in a complementary strand (the secondary strand). The N-terminal zinc finger of the three-zinc-finger domain of Sp1 has inherently a unique five-base-pair binding mode in which the guanine bases are recognized in both strands. To clarify the effect of the amino acid at position 2 on DNA binding affinity and base specificity, we have created a library of the mutants by the interconversion between serine and aspartic acid in the N-terminal zinc finger of Sp1 and recombinant variants of finger order. Gel mobility shift and methylation interference assays showed that the combination of arginine and serine at positions -1 and 2, respectively, provides a newly strong guanine contact in the secondary strand and a higher binding affinity than that of wild-type Sp1. Of special interest are the facts that the mutant with lysine and aspartic acid at positions -1 and 2 in the alpha helix predominantly recognizes the bases in the secondary strand and that its DNA binding affinity is higher than that of the wild-type. The aspartic acid or serine at position 2 independently contributes to the DNA binding affinity and base specificity. The present results provide useful information for the design of a novel zinc finger protein with priority for the bases in the secondary strand.     

Plant aspartic proteinases: enzymes on the way to a function

Plant aspartic proteinases have been characterized from seeds, flowers and leaves of a number of different species. The enzymes are generally either monomeric or heterodimeric, containing two peptides processed from the same precursor protein. The plant enzymes, like their mammalian and microbial counterparts, are active at acidic pH and inhibited by a class specific inhibitor pepstatin A. Plant aspartic proteinases are generally either secreted or targeted to the vacuolar/protein storage body compartment. The primary sequences of many of these enzymes have been determined and are very homologous with each other as well as with enzymes from mammalian and microbial origins. Plant aspartic proteinases, however, have a very unique plant specific region, which is not found in mammalian, microbial, or viral aspartic proteinases. The function of this region has not been elucidated. A role for these plant enzymes in protein processing or degradation has been proposed, however, more studies are required to confirm their in vivo functions. Recent intriguing results suggest possible roles for these enzymes in programmed cell-death of tissues and in pathogen resistance.     

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion

Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here, we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans. A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H. americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen.     

Purification and characterization of aspartic proteinase from sunflower seeds

Aspartic proteinases were purified from sunflower seed extracts by affinity chromatography on a pepstatin A-EAH Sepharose column and by Mono Q column chromatography. The final preparation contained three purified fractions. SDS-PAGE showed that one of the fractions consisted of disulfide-bonded subunits (29 and 9 kDa), and the other two fractions contained noncovalently bound subunits (29 and 9 kDa). These purified enzymes showed optimum pH for hemoglobinolytic activity at pH 3.0 and were completely inhibited by pepstatin A like other typical aspartic proteinases. Sunflower enzymes showed more restricted specificity on oxidized insulin B chain and glucagon than other aspartic proteinases. The cDNA coding for an aspartic proteinase was cloned and sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 440 amino acid residues with a molecular mass of 47,559 Da. The difference between the molecular size of purified enzymes and of the mature enzyme was due to the fact that the purified enzymes were heterodimers formed by the proteolytic processing of the mature enzyme. The derived amino acid sequence of the enzyme showed 30-78% sequence identity with that of other aspartic proteinases.     

Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii

Aspartic proteinases have been extensively characterized in land plants but up to now no evidences for their presence in green algae group have yet been reported in literature. Here we report on the identification of the first (and only) typical aspartic proteinase from Chlamydomonas reinhardtii. This enzyme, named chlapsin, was shown to maintain the primary structure organization of typical plant aspartic proteinases but comprising distinct features, such as similar catalytic motifs DTG/DTG resembling those from animal and microbial counterparts, and an unprecedentedly longer plant specific insert domain with an extra segment of 80 amino acids, rich in alanine residues. Our results also demonstrated that chlapsin accumulates in Chlamydomonas chloroplast bringing this new enzyme to a level of uniqueness among typical plant aspartic proteinases. Chlapsin was successfully expressed in Escherichia coli and it displayed the characteristic enzymatic properties of typical aspartic proteinases, like optimum activity at acidic pH and complete inhibition by pepstatin A. Another difference to plant aspartic proteinases emerged as chlapsin was produced in an active form without its putative prosegment domain. Moreover, recombinant chlapsin showed a restricted enzymatic specificity and a proteolytic activity influenced by the presence of redox agents and nucleotides, further differentiating it from typical plant aspartic proteinases and anticipating a more specialized/regulated function for this Chlamydomonas enzyme. Taken together, our results revealed a pattern of complexity for typical plant aspartic proteinases in what concerns sequence features, localization and biochemical properties, raising new questions on the evolution and function of this vast group of plant enzymes.