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PubMed2002

Structure of human nicotinamide/nicotinic acid mononucleotide adenylyltransferase. Basis for the dual substrate specificity and activation of the oncolytic agent tiazofurin.

Nicotinamide/nicotinate mononucleotide (NMN/ NaMN)adenylyltransferase (NMNAT) is an indispensable enzyme in the biosynthesis of NAD(+) and NADP(+). Human NMNAT displays unique dual substrate specificity toward both NMN and NaMN, thus flexible in participating in both de novo and salvage pathways of NAD synthesis. Human NMNAT also catalyzes the rate-limiting step of the metabolic conversion of the anticancer agent tiazofurin to its active form tiazofurin adenine dinucleotide (TAD). The tiazofurin resistance is mainly associated with the low NMNAT activity in the cell. We have solved the crystal structures of human NMNAT in complex with NAD, deamido-NAD, and a non-hydrolyzable TAD analogue beta-CH(2)-TAD. These complex structures delineate the broad substrate specificity of the enzyme toward both NMN and NaMN and reveal the structural mechanism for adenylation of tiazofurin nucleotide. The crystal structure of human NMNAT also shows that it forms a barrel-like hexamer with the predicted nuclear localization signal sequence located on the outside surface of the barrel, supporting its functional role of interacting with the nuclear transporting proteins. The results from the analytical ultracentrifugation studies are consistent with the formation of a hexamer in solution under certain conditions.

Amino Acid Sequence↗
PubMed2005

Secretion of YscP from Yersinia enterocolitica is essential to control the length of the injectisome needle but not to change the type III secretion substrate specificity.

The length of the needle of the Yersinia Ysc injectisome is determined by a protein called YscP. This protein, which acts both as a molecular ruler and as a substrate-specificity switch for type III secretion is itself secreted by the injectisome. In this report, we address the question why YscP is secreted. By a systematic deletion analysis and by fusing different parts of the molecule to the adenylate cyclase reporter, we identified two independent secretion signals. One of them is encompassed within the 35 N-terminal residues while the second one spans residues 97-137. These two signals are functionally different from Yop secretion signals. When both secretion signals were removed, Yops could still be secreted but the needle length control was lost. YscP possessing only one signal did not control needle length properly but the control was improved when more YscP was produced and secreted. YscP deprived of both signals could not control length, even when overproduced. We conclude from this that YscP needs to be secreted to exert its length control function but not its substrate-specificity switch function.

Bacterial Proteins↗
PubMed1986

Substrate specificities of tyrosine-specific protein kinases toward cytoskeletal proteins in vitro.

We have previously reported that fodrin (beta subunit), tubulin (alpha subunit) and microtubule-associated proteins (MAPs; MAP2 and tau) are good substrates for the purified insulin receptor kinase (Kadowaki, T., Nishida, E., Kasuga, M., Akiyama, T., Takaku, F., Ishikawa, M., Sakai, H., Kathuria, S., and Fujita-Yamaguchi, Y. (1985) Biochem. Biophys. Res. Commun. 127, 493-500 and Kadowaki, T., Fujita-Yamaguchi, Y., Nishida, E., Takaku, F., Akiyama, T., Kathuria, S., Akanuma, Y., and Kasuga, M. (1985) J. Biol. Chem. 260, 4016-4020). In this study, to investigate the substrate specificities of tyrosine kinases, we have examined the actions of the purified epidermal growth factor (EGF) receptor kinase and Rous sarcoma virus src kinase on purified microfilament- and microtubule-related proteins. Among microfilament-related proteins examined, the purified EGF receptor kinase phosphorylated the beta subunit, but not the alpha subunit, of fodrin on tyrosine residues with a Km below the micromolar range. The fodrin phosphorylation by the EGF receptor kinase was markedly inhibited by F-actin. In contrast, the purified src kinase preferentially phosphorylated the alpha subunit of fodrin on tyrosine residues. Fodrin phosphorylation by the src kinase was not inhibited by F-actin. Among microtubule proteins examined, MAP2 was the best substrate for the EGF receptor kinase. By contrast, src kinase favored phosphorylation of tubulin as compared to MAP2. The peptide mapping of MAP2 phosphorylated by the EGF receptor kinase and by the insulin receptor kinase produced very similar patterns of phosphopeptides, while that of MAP2 phosphorylated by the src kinase gave a distinctly different pattern. When the phosphorylation of the tubulin subunits was examined, the EGF receptor kinase preferred beta subunit to alpha subunit, but the src kinase phosphorylated both alpha and beta subunits to a similar extent. These results, together with our previous results, indicate that the substrate specificities of the EGF receptor kinase and the insulin receptor kinase are very similar, but not identical, while that of the src kinase is distinctly different from that of these growth factor receptor kinases.

Amino Acids↗
PubMed2005

Fusion-based strategies to identify genes involved in degradation of a specific substrate.

Fusion proteins have been used in many instances to allow genetic screening for genes required for the degradation of a specific substrate. This straightforward, yet powerful, approach can be applied in many circumstances to facilitate gene characterization and discovery. Some general principles are discussed and then several successful uses of these tactics are described in detail.

Gene Fusion↗
PubMed1998

The Epstein-Barr virus thymidine kinase does not phosphorylate ganciclovir or acyclovir and demonstrates a narrow substrate specificity compared to the herpes simplex virus type 1 thymidine kinase.

The Epstein-Barr virus (EBV) thymidine kinase (TK) was expressed in mammalian 143B TK- cells to investigate its substrate specificity. The herpes simplex virus type 1 (HSV-1) TK was similarly expressed for comparison. Both viral TKs conferred a TK+ phenotype on 143B TK- cells. The nucleoside analog ganciclovir (GCV) did not affect the growth of 143B EBV TK or 143B TK- cells but effectively killed 143B HSV-1 TK cells. Furthermore, lysates of 143B EBV TK cells could not phosphorylate GCV, which was confirmed by high-performance liquid chromatography. EBV TK, HSV-1 TK, and EBV TK N-, a truncated EBV TK missing 243 N-terminal amino acids, were purified as fusion proteins expressed in bacteria, and all had TK activity. In addition, EBV TK was observed to have a thymidylate kinase activity but could not phosphorylate GCV, acyclovir, or 2'-deoxycytidine. In competition assays, only nucleoside analogs of thymidine significantly inhibited thymidine phosphorylation by EBV TK, with the following rank order: 5-bromodeoxyuridine > zidovudine > stavudine > sorivudine. These results demonstrate that EBV TK substrate specificity is narrower than those of alphaherpesvirus TKs and that thymidine analogs may be the most suitable nucleoside antivirals to target the enzyme. Clinical implications for gammaherpesviruses are discussed.

Acyclovir↗
PubMed1991

Differences in substrate specificities of monoamine oxidase A from human liver and placenta.

The substrate specificities of monoamine oxidase (MAO) A isolated from human placenta and of human liver expressed in yeast have been compared in homogeneous preparations with respect to Vmax and Km values for natural and synthetic substrates and Ki values for competitive inhibitors. MAO A from these two sources is known to differ in at least 5 amino acid residues. While the Km and Ki values were found to be nearly identical in the enzymes from these two sources, the Vmax differed significantly on bulky synthetic substrates.

1-Methyl-4-phenylpyridinium↗
PubMed2005

Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals.

The P450 cytochromes constitute a large family of hemoproteins that catalyze the monooxygenation of a diversity of hydrophobic substrates. CYP102A2 is a catalytically self-sufficient cytoplasmic enzyme from Bacillus subtilis, containing both a monooxygenase domain and a reductase domain on a single polypeptide chain. CYP102A2 was subjected to error-prone PCR to generate mutants with enhanced activity with fatty acids and other aromatic substrates. The library of CYP102A2 mutants was expressed in BL21(DE3) Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. After a single round of error-prone PCR, the variant Pro15Ser exhibiting modified substrate specificity was generated. This variant showed approximately 6- to 9-fold increased activity with SDS, lauric acid and 1,4-naphthoquinone, and enhanced activity for other substrates such as ethacrynic acid and epsilon-amino-n-caproic acid. Molecular modeling of the CYP102A2 monooxygenase domain suggested that Pro15 is located in a short helical segment and is involved in extensive interactions between the N-terminal domain and the beta2 sheet, which contribute to the formation of the substrate binding site. Thus, Pro15 appears to affect substrate binding and catalysis indirectly. These results clearly demonstrate the importance of remote residues, not readily predicted by rational design, for the determination of substrate specificity. In addition, we report here that the Pro15Ser variant of CYP102A2 can be efficiently immobilized on epoxy-activated Sepharose at pH 8.5 and 4 degrees C. The immobilized variant of CYP102A2 retains most of its activity (81%) and shows improved stability at 37 degrees C. The approach offers the possibility of designing a P450 bioreactor that can be operated over a long period of time with high efficiency and which can be used in fine chemical synthesis.

Amino Acid Sequence↗
PubMed2005

Defining the substrate specificity of mouse cathepsin P.

Cathepsin P is a recently discovered placental cysteine protease that is structurally related to the more ubiquitously expressed, broad-specificity enzyme, cathepsin L. We studied the substrate specificity requirements of recombinant mouse cathepsin P using fluorescence resonance energy transfer (FRET) peptides derived from the lead sequence Abz-KLRSSKQ-EDDnp (Abz, ortho-aminobenzoic acid and EDDnp, N-[2,4-dinitrophenyl]ethylenediamine). Systematic modifications were introduced resulting in five series of peptides to map the S(3) to S(2)(') subsites of the enzyme. The results indicate that the subsites S(1), S(2), S(1)('), and S(2)('), present a clear preference for hydrophobic residues. The specificity requirements of the S(2) subsite were found to be more restricted, preferring hydrophobic aliphatic amino acids. The S(3) subsite of the enzyme presents a broad specificity, accepting negatively charged (Glu), positively charged (Lys, Arg), and hydrophobic aliphatic or aromatic residues (Val, Phe). For several substrates, the activity of cathepsin P was markedly regulated by kosmotropic salts, particularly Na(2)SO(4). No significant effect on secondary or tertiary structure could be detected by either circular dichroism or size exclusion chromatography, indicating that the salts most probably disrupt unfavorable ionic interactions between the substrate and enzyme active site. A substrate based upon the preferred P(3) to P(2)(') defined by the screening study, ortho-aminobenzoic-Glu-Ile-Phe-Val-Phe-Lys-Gln-N-(2,4-dinitrophenyl)ethylenediamine (cleaved at the Phe-Val bond) was efficiently hydrolyzed in the absence of high salt. The k(cat)/K(m) for this substrate was almost two orders of magnitude higher than that of the original parent compound. These results show that cathepsin P, in contrast to other mammalian cathepsins, has a restricted catalytic specificity.

Animals↗
PubMed1999

Towards regioselective synthesis of oligosaccharides by use of alpha-glucosidases with different substrate specificity.

alpha-Glucosidase from two microbial sources, Bacillus stearothermophilus and Brewer's yeast, has been used to catalyze transglycosylation reactions and a comparative study was carried out to determine the regioselectivity of this reaction. Bacterial alpha-glucosidase exhibited higher transfer activity with maltose and was able to synthesize tri- and tetrasaccharides in high yield (27%). In the case of yeast enzyme, only trisaccharides were synthesized in lower yield. Structure analysis of transglycosylation products by means of GC-MS and NMR spectroscopy revealed a correlation between the hydrolytic substrate specificity and the regioselectivity of transglycosylation reaction. Higher substrate specificity of bacterial enzyme, however, influenced its transglucosylation activity toward other saccharide acceptors.

Chromatography, High Pressure Liquid↗
PubMed2005

Using quantitative imaging microscopy to define the target substrate specificities of histone post-translational-modifying enzymes.

Until recently, identifying the specificities of enzymes that post-translationally modify core histones was performed in vitro using synthetic peptides, purified mononucleosomes or short nucleosome arrays. Unfortunately, the variable results obtained for identical enzymes are often dependent on the in vitro conditions employed. These results are consistent with the conclusion that the manner in which histone tails are presented to the modifying enzymes dramatically affects specificity. Because traditional in vitro biochemical approaches do not accurately recapitulate higher-order chromatin structure or consider the influences that additional chromatin binding proteins may have on determining the specificity of modifying enzymes, the development of new and innovative approaches is warranted. Here, we describe a novel in situ microscopy approach that accurately assesses enzyme substrate specificities through single cell measurements performed under physiologically relevant conditions. This approach couples the spatial resolving power of microscopy with robust statistical analyses to determine the substrate specificities of transiently expressed enzymes using histone modification- and residue-specific antibodies. This methodology can also be applied to measuring changes in the abundance of histone modifications as cells traverse the cell cycle.

Acetylation↗
PubMed1997

Functional expression of a cDNA to human acyl-coenzyme A:cholesterol acyltransferase in yeast. Species-dependent substrate specificity and inhibitor sensitivity.

We have identified two yeast genes with similarity to a human cDNA encoding acyl-coenzyme A:cholesterol acyltransferase (ACAT). Deletion of both yeast genes results in a viable cell with undetectable esterified sterol (Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R. J., Aljinovic, G., Pohl, T., Rothstein, R., and Sturley, S. L. (1996) Science 272, 1353-1356). Here, we expressed the human cDNA in the yeast double mutant, resulting in high level production of ACAT protein, but low in vivo esterification of ergosterol, the predominant yeast sterol. The activity of the human enzyme was increased by incubation of these cells with 25-hydroxy, cholesterol, an established positive regulator of mammalian sterol esterification. In contrast, the yeast enzymes were unaffected by this reagent. In vitro microsomal assays indicated no sterol esterification in extracts from the double mutant. However, significant activity was detected from strains expressing human ACAT when cholesterol was equilibrated with the microsomal membranes. The human enzyme in yeast utilized cholesterol as the preferred sterol and was sensitive to competitive (S58035) and non-competitive (DuP 128) ACAT inhibitors. The yeast esterifying enzymes exhibited a diminished sterol substrate preference and were sensitive only to S58035. Human ACAT had a broad acyl-CoA substrate specificity, the other substrate for this reaction. By contrast, the yeast enzymes had a marked preference for specific acyl-CoAs, particularly unsaturated C18 forms. These results confirm the yeast genes as functional homologs of the human gene and demonstrate that the enzymes confer substrate specificity to the esterification reaction in both organisms.

Cholesterol↗
PubMed1994

Kinetics of the two forms of acetyl-CoA carboxylase from Pisum sativum. Correlation of the substrate specificity of the enzymes and sensitivity towards aryloxyphenoxypropionate herbicides.

Steady-state kinetics of the 220-kDa form of acetyl-CoA carboxylase (ACC220), as purified from mature pea seeds, have been investigated with respect to the substrate specificity and inhibition by quizalofop, a herbicide of the aryloxyphenoxypropionate type. The enzyme showed a dual specificity, being able to carboxylate propionyl-CoA at a maximal rate approximately 20% that measured in the presence of the acetyl-CoA substrate. These two reactions occur at separate sites on the enzyme. One site binds either acetyl-CoA (Km = 226 microM) or propionyl-CoA (Km = 38 microM) and is strongly inhibited by quizalofop (Ki = 25 microM and 9.3 microM for the acetyl-CoA and propionyl-CoA substrates, respectively). The other is specific for acetyl-CoA (Km = 11 microM) and is much less inhibited by quizalofop (Ki = 256 microM). Owing to the existence of these two catalytically different sites, the enzyme obeyed Michaelis-Menten kinetics with propionyl-CoA, but exhibited kinetic co-operativity in the presence of acetyl-CoA. Also, kinetics of propionyl-CoA carboxylase activity of ACC220 exhibited hyperbolic inhibition in the presence of quizalofop, but co-operative inhibition when following the ACC activity of the enzyme. The results suggest that the higher the substrate specificity, the lower the quizalofop sensitivity of the active site. Similar kinetic behaviour was observed with ACC220 purified from pea leaves. Also, the apparent correlation between the substrate specificity and the sensitivity of ACC towards quizalofop was confirmed by kinetic analyses of the low-molecular-mass form of ACC present in chloroplasts of young pea leaves. This enzyme was insensitive to quizalofop inhibition and was not able to carboxylate propionyl-CoA. No other propionyl-CoA carboxylase activity, different from that catalysed by ACC220, could be detected from either reproductive or vegetative organs of pea plants at any stage of development.

Acetyl Coenzyme A↗
PubMed1989

Kinetic investigation of the substrate specificity of the cyanogenic beta-D-glucosidase (linamarase) of white clover.

Partially purified linamarase from Trifolium repens (genotype Lili acac) plants was kinetically characterized. Kinetic evidence was found to support the assumption that this cyanogenic beta-D-glucosidase has a broad substrate spectrum. p-Nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-alpha-L-arabinopyranoside substrates bound almost as tightly to the active center of the enzyme as the glucono(1----5)lactone transition-state analog inhibitor. Substrate specificity investigation also indicated that positions C-4 and C-6 on the pyranoside ring play an essential role in both substrate orientation and splitting. Recently very similar kinetic characteristics were reported on some mammalian cytosolic beta-D-glucosidases and a possible physiological interpretation of this coincidence is discussed. Inhibition studies with glucono(1----5)lactone revealed that the carbohydrate moiety of each substrate attached to the same binding site in the active center. Inhibition experiments with 1-thio substrate analogs demonstrated that the aglycon and the angular arrangement around the glycosidic linkage were the major determinants in the observed substrate specificity.

Binding, Competitive↗
PubMed2003

The oligomeric structure of human granzyme A is a determinant of its extended substrate specificity.

The cell death-inducing serine protease granzyme A (GzmA) has a unique disulfide-linked quaternary structure. The structure of human GzmA bound to a tripeptide CMK inhibitor, determined at a resolution of 2.4 A, reveals that the oligomeric state contributes to substrate selection by limiting access to the active site for potential macromolecular substrates and inhibitors. Unlike other serine proteases, tetrapeptide substrate preferences do not correlate well with natural substrate cleavage sequences. This suggests that the context of the cleavage sequence within a macromolecular substrate imposes another level of selection not observed with the peptide substrates. Modeling of inhibitors bound to the GzmA active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft. The crystal structure, along with substrate library profiling and mutagenesis, has allowed us to identify and rationally manipulate key components involved in GzmA substrate specificity.

Amino Acid Sequence↗
PubMed1998

Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity.

Cytochrome P-450 CYP2D6, human debrisoquine hydroxylase, metabolizes more than 30 prescribed drugs, the vast majority of which are small molecules containing a basic nitrogen atom. In contrast, the similar mouse protein Cyp2d-9 was first characterized as a testosterone 16alpha-hydroxylase. No common substrates have been reported for the two enzymes. Here we investigate the structural basis of this difference in substrate specificity. We have earlier used a combination of NMR data and homology modelling to generate a three-dimensional model of CYP2D6 [Modi, Paine, Sutcliffe, Lian, Primrose, Wolf, C.R. and Roberts (1996) Biochemistry 35, 4541-4550]. We have now generated a homology model of Cyp2d-9 and compared the two models to identify specific amino acid residues that we believe form the substrate-binding site in each protein and therefore influence catalytic selectivity. Although there are many similarities in active site structure, the most notable difference is a phenylalanine residue (Phe-483) in CYP2D6, which in the model is located such that the bulky phenyl ring is positioned across the channel mouth, thus limiting the size of substrate that can access the active site. In Cyp2d-9, the corresponding position is occupied by an isoleucine residue, which imposes fewer steric restraints on the size of substrate that can access the active site. To investigate whether the amino acid residue at this position does indeed influence the catalytic selectivity of these enzymes, site-directed mutagenesis was used to change Phe-483 in CYP2D6 to isoleucine and also to tryptophan. CYP2D6, Cyp2d-9 and both mutant CYP2D6 proteins were co-expressed with NADPH cytochrome P-450 reductase as a functional mono-oxygenase system in Escherichia coli and their relative catalytic activities towards bufuralol and testosterone were determined. All four proteins exhibited catalytic activity towards bufuralol but only Cyp2d-9 catalysed the formation of 16alpha-hydroxytesterone. Uniquely, the CYP2D6F483I mutant acquired the ability to metabolize testosterone to a novel product, which was identified by MS and proton NMR spectroscopy as 15alpha-hydroxytestosterone. NMR spin relaxation experiments were used to measure distances between the haem iron and protons of testosterone bound to the CYP2D6F483I mutant. These experiments demonstrate that very minor modifications to the active site structure of CYP2D6 can have a profound influence on the substrate specificity of the enzyme.

Amino Acid Sequence↗
PubMed1991

Limited proteolysis of rat brain tyrosine hydroxylase defines an N-terminal region required for regulation of cofactor binding and directing substrate specificity.

Trypsin proteolysis of tyrosine hydroxylase (TH) produces a 34-kDa fragment that is catalytically active but does not contain the regulatory phosphorylation sites. In this report, activation of TH by proteolysis was characterized further. Proteolysis results in a decrease in Kms for both substrate and cofactor. The increase in affinity for cofactor was identical to that produced by phosphorylation with cAMP-dependent protein kinase. Additionally, proteolysis of an N-terminal region containing the regulatory phosphorylation sites was sufficient to produce a decrease in Km for cofactor. Activation of substrate binding required more extensive proteolysis but also corresponded to N-terminal digestion. Moreover, this activation was coincident with a broadened substrate specificity. In combination, these data indicate that the N-terminus of tyrosine hydroxylase regulates cofactor binding and directs substrate specificity.

Animals↗
PubMedDate not supplied

[Substrate specificity of brain cholinesterase activity of evolutionary old species of fishes].

An analysis of brain cholinesterase properties in 3 species of cartilaginous fishes and 1 species of chondrostei, was conducted for the first time. The skates brain enzymes, as to substrate specificity, are shown to be referred to acetylcholinesterases, and the shark brain cholinesterase - to atypical acetylcholinesterase. Kaluga brain cholinesterase, as to its substrate specificity, differes from brain enzymes of cartilaginous fishes. The differences in properties of the nervous tissue cholinestarase of the investigated fish species are the parameters of evolutionary divergence of species.

Acetylcholinesterase↗
PubMed2006

Substrate specificity of inner membrane peptidase in yeast mitochondria.

The inner membrane protease (IMP) cleaves intra-organelle sorting peptides from precursor proteins in mitochondria of the yeast Saccharomyces cerevisiae. An unusual feature of the IMP is the presence of two catalytic subunits, Imp1p and Imp2p, which recognize distinct substrate sets even though both enzymes belong to the same protease family. This nonoverlapping substrate specificity was hypothesized to result from the recognition of distinct residues at the P'1 position (also termed +1 position) in the protease substrates. Here, we constructed an extensive series of mutations to obtain a profile of the critical cleavage site residues in IMP substrates and conclude that Imp1p, and not Imp2p, recognizes specific P'1 residues. In addition to its specificity for P'1 residues, Imp1p also shows substrate specificity for the P3 (-3) position. In contrast, Imp2p recognizes the P1 (-1) position and the P3 position. Based on this new understanding of IMP substrate specificity, we conducted a survey for candidate IMP substrates in mammalian mitochondria and found consensus Imp2p cleavage sites in mammalian precursors to cytochrome c1 and glycerol-3-phosphate (G-3-P) dehydrogenase. Presence of a putative Imp2p cleavage site in G-3-P dehydrogenase was surprising, as its yeast ortholog contains an Imp1p cleavage site. To address this issue experimentally, we performed the first co-expression of mammalian IMP with proposed mammalian IMP precursors in yeast and show that murine precursors to cytochrome c1 and G-3-P dehydrogenase are cleaved by murine Imp2p. These results suggest, surprisingly, G-3-P dehydrogenase has switched from Imp1p in yeast to Imp2p in mammals.

Animals↗