Interactions at the dimer interface influence the relative efficiencies for purine nucleotide synthesis and pyrophosphorolysis in a phosphoribosyltransferase

Enzymes that salvage 6-oxopurines, including hypoxanthine phosphoribosyltransferases (HPRTs), are potential targets for drugs in the treatment of diseases caused by protozoan parasites. For this reason, a number of high-resolution X-ray crystal structures of the HPRTs from protozoa have been reported. Although these structures did not reveal why HPRTs need to form dimers for catalysis, they revealed the existence of potentially relevant interactions involving residues in a loop of amino acid residues adjacent to the dimer interface, but the contributions of these interactions to catalysis remained poorly understood. The loop, referred to as active-site loop I, contains an unusual non-proline cis-peptide and is composed of residues that are structurally analogous with Leu67, Lys68, and Gly69 in the human HPRT. Functional analyses of site-directed mutations (K68D, K68E, K68N, K68P, and K68R) in the HPRT from Trypanosoma cruzi, etiologic agent of Chagas' disease, show that the side-chain at position 68 can differentially influence the K(m) values for all four substrates as well as the k(cat) values for both IMP formation and pyrophosphorolysis. Also, the results for the K68P mutant are inconsistent with a cis-trans peptide isomerization-assisted catalytic mechanism. These data, together with the results of structural studies of the K68R mutant, reveal that the side-chain of residue 68 does not participate directly in reaction chemistry, but it strongly influences the relative efficiencies for IMP formation and pyrophosphorolysis, and the prevalence of lysine at position 68 in the HPRT of the majority of eukaryotes is consistent with there being a biological role for nucleotide pyrophosphorolysis.     

Saturation mutagenesis, complement selection, and steady-state kinetic studies illuminate the roles of invariant residues in active site loop I of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi

Hypoxanthine phosphoribosyltransferases (HPRTs) are potential drug targets in the treatment of diseases caused by parasites. Also, defects in the human HPRT can result in gouty arthritis or Lesch-Nyhan syndrome. Active site loop I of HPRTs has been implicated in interactions between enzyme subunits that can influence the relative efficiencies of forward and reverse reactions, but the functional roles for invariant loop I residues (analogous with human Leu67 and Gly69) are poorly understood. Herein, saturation mutagenesis, complement selection, and steady-state kinetics were used to investigate the functional roles for Leu67 and Gly69. Seventy clones from a library of mutants were sequenced and more than 30 different mutations, or combinations of mutations, were identified. Several recombinant HPRTs with mutations at positions 67 and/or 69 supported the growth of a bacterial auxotroph on selective media, but only two of the mutants (L67M and G69S) could be recovered in the soluble fraction from bacteria induced to over-express the enzyme. The results of steady-state kinetic studies for L67M are consistent with the side chain of this residue participating in hydrophobic interactions between dimer subunits that are important for the proper positioning of main chain atoms that influence enzyme chemistry and the binding of PRPP, PPi, and hypoxanthine. The results for mutations at position 69 are consistent with only hydrogen or a small polar side chain being tolerated at this site. Kinetic studies of G69S suggest that side chains of residues at position 69 that project into the active site likely interfere with the binding of PRPP and PPi, as well as the positioning of a metal ion that indirectly influences the binding of purine bases and purine moieties of nucleotide substrates.     

Mechanisms of allosteric regulation of Trypanosoma cruzi S-adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the k(cat)/K(m) for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC(50)s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 microM), while at higher concentrations (>100 microM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.     

Replacement by site-directed mutagenesis indicates a role for histidine 170 in the glutamine amide transfer function of anthranilate synthase

Anthranilate synthase is a glutamine amidotransferase that catalyzes the first reaction in tryptophan biosynthesis. Conserved amino acid residues likely to be essential for glutamine-dependent activity were identified by alignment of the glutamine amide transfer domains in four different enzymes: anthranilate synthase component II (AS II), p-aminobenzoate synthase component II, GMP synthetase, and carbamoyl-P synthetase. Conserved amino acids were mainly localized in three clusters. A single conserved histidine, AS II His-170, was replaced by tyrosine using site-directed mutagenesis. Glutamine-dependent enzyme activity was undetectable in the Tyr-170 mutant, whereas the NH3-dependent activity was unchanged. Affinity labeling of AS II active site Cys-84 by 6-diazo-5-oxonorleucine was used to distinguish whether His-170 has a role in formation or in breakdown of the covalent glutaminyl-Cys-84 intermediate. The data favor the interpretation that His-170 functions as a general base to promote glutaminylation of Cys-84. Reversion analysis was consistent with a proposed role of His-170 in catalysis as opposed to a structural function. These experiments demonstrate the application of combining sequence analyses to identify conserved, possibly functional amino acids, site-directed mutagenesis to replace candidate amino acids, and protein chemistry for analysis of mutationally altered proteins, a regimen that can provide new insights into enzyme function.     

Directed evolution of rubisco in Escherichia coli reveals a specificity-determining hydrogen bond in the form II enzyme

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) occupies a critical position in photosynthetic CO2-fixation and consequently has been the focus of intense study. Crystal-structure-guided site-directed mutagenesis studies have met with limited success in engineering kinetic improvements to Rubisco, highlighting our inadequate understanding of structural constraints at the atomic level that dictate the enzyme's catalytic chemistry. Bioselection provides an alternative random mutagenic approach that is useful for identifying and elucidating imperceptible structure-function relationships. Using the dimeric Form II Rubisco from Rhodospirillum rubrum, its gene (rbcM) was randomly mutated and introduced under positive selection into Escherichia coli cells metabolically engineered to be dependent on Rubisco to detoxify its substrate ribulose 1,5-bisphosphate. Thirteen colonies displaying improved fitness were isolated, and all were found to harbor mutations in rbcM at one of two codons, histidine-44 or aspartate-117, that are structurally adjacent amino acids located about 10 A from the active site. Biochemical characterization of the mutant enzymes showed the mutations reduced their CO2/O2 specificity by 40% and decreased their carboxylation turnover rate by 20-40%. Structural analyses showed histidine-44 and aspartate-117 form a hydrogen bond in R. rubrum Rubisco and that the residues are conserved among other Form II Rubiscos. This study demonstrated the utility of directed evolution in E. coli for identifying catalytically relevant residues (in particular nonobvious residues disconnected from active site residues) and their potential molecular interactions that influence Rubisco's catalytic chemistry.     

Cleavage of type I and type II procollagens by type I/II procollagen N-proteinase. Correlation of kinetic constants with the predicted conformations of procollagen substrates

The kinetic constants were examined for the cleavage of several types of procollagen by type I/II procollagen N-proteinase. The Km values were essentially the same (0.2 microM) for chick type I procollagen, human type I procollagen, and chick type II procollagen. However, the Vmax values differed over a 14-fold range. As reported previously, the enzyme did not cleave denatured type I or II procollagen. Also, it did not cleave human type III procollagen which contains the same scissle -Pro-Gln- bond as the pro-alpha 1(I) chain of type I procollagen. To explain the observations, Chou-Fasman rules were used to compare the secondary structures of the cleavage sites in the procollagens. The results supported a previous suggestion (Helseth, D. L., Jr., Lechner, J. L., and Veis, A. (1979) Biopolymers 18, 3005-3014) that the region carboxyl-terminal to cleavage site in the pro-alpha 1(I) chain of type I procollagen was in a hairpin conformation consisting of a beta-sheet, beta-turn, and beta-sheet. In both chick and human type I procollagen, the hairpin loop in the pro-alpha 1(I) chain consisted of about 18 amino acids. The cleavage site itself was in a short alpha-helical structure of four or five amino acids. The pro-alpha 2(I) chains had a similar hairpin loop of about 14 amino acids and alpha-helix of four or five amino acids containing the cleavage site. Chick type II procollagen, which had the highest Vmax value, had a longer hairpin structure of 22 amino acids, and the cleavage site was in a longer alpha-helical domain of 10 amino acids. In contrast, type III procollagen had a random-coil conformation in the same region. The results help to explain the unusual substrate requirements of type I/II N-proteinase. They also help explain why mutations that produce in-frame deletions of amino acids 84 or more residues carboxyl-terminal to the cleavage site make the protein resistant to the enzyme.     

Identification of 17 independent mutations responsible for human hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency.

Complete hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency causes the Lesch-Nyhan syndrome, an X-linked, purine metabolism disorder manifested by hyperuricemia, hyperuricaciduria, and neurologic dysfunction. Partial HPRT deficiency causes hyperuricemia and gout. One requirement for understanding the molecular basis of HPRT deficiency is the determination of which amino acids in this salvage enzyme are necessary for structural or catalytic competence. In this study we have used the PCR coupled with direct sequencing to determine the nucleotide and subsequent amino acid changes in 22 subjects representing 17 unrelated kindreds from the United Kingdom. These mutations were confirmed by using either RNase mapping or Southern analyses. In addition, experiments were done to determine enzyme activity and electrophoretic mobility, and predictive paradigms were used to study the impact of these amino acid substitutions on secondary structure.     

Identification of the active conformation and the importance of length of the flexible loop 72-83 in regulating the conformational change of undecaprenyl pyrophosphate synthase

Increasing evidence has shown that intrinsic disorder of proteins plays a key role in their biological functions. In the case of undecaprenyl pyrophosphate synthase (UPPs), which catalyzes the chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate via eight consecutive condensation reactions with isopentenyl pyrophosphate, a highly flexible loop 72-83 was previously linked to protein conformational change required for catalysis [Chen, Y. H., Chen, A. P.-C., Chen, C. T., Wang, A. H.-J., and Liang, P. H., (2002) J. Biol. Chem. 277, 7369-7376]. The crystal structure and fluorescence studies suggested that the alpha3 helix connected to the loop moves toward the active site when the substrate is bound. To identify the active conformation and study the role of the loop for conformational change, the UPPs mutants with amino acids inserted into or deleted from the loop were examined. The inserted mutant with extra Ala residues fails to display the intrinsic fluorescence quenching upon FPP binding, and its crystal structure reveals only the open form. These phenomena appear to be different from the wild-type enzyme in which open and closed conformers were observed and suggest that the extended loop fails to pull the alpha3 helix and/or the extra amino acids in the loop cause steric hindrance on the alpha3 helix movement. The loop-shortening mutants with deletion of V82 and S83 or S72 also adopt an open conformation with the loop stretched, although they show decreased intrinsic fluorescence with FPP bound, similar to that seen in the wild-type enzyme. We conclude that the closed conformation is apparently the active conformation. Change of the length of the loop 72-83 impairs the ability of conformational change and causes remarkably lower activity of UPPs.     

Sequence of cloned enzyme IIN-acetylglucosamine of the phosphoenolpyruvate:N-acetylglucosamine phosphotransferase system of Escherichia coli

In Escherichia coli, N-acetylglucosamine (nag) metabolism is joined to glycolysis via three specific enzymes that are the products of the nag operon. The three genes of the operon, nagA, nagB, and nagE, were found to be carried by a colicin plasmid, pLC5-21, from a genomic library of E. coli [Clarke, L., & Carbon, J. (1976) Cell (Cambridge, Mass.) 9,91-99]. The nagE gene that codes for enzyme IIN-acetylglucosamine of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was sequenced. The nagE sequence is preceded by a catabolite gene activator protein binding site and ends in a putative rho-independent termination site. The amino acid sequence determined from this DNA sequence shows 44% homology to enzymes IIglucose and IIIglucose of the PTS. Enzyme IIN-acetylglucosamine, which has 648 amino acids and a molecular weight of 68,356, contains a histidine at residue 569 which is homologous to the active site of IIIglc. Sequence homologies with enzymes IIglucose, II beta-glucoside, and IIsucrose indicate that residues His-190, His-213, and His-295 of enzyme IInag are also conserved and that His-190 is probably the second active site histidine. Other sequence homologies among these enzymes II suggest that they contain several sequence transpositions. Preliminary models of the enzymes II are proposed.     

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

Improvement in thermostability and psychrophilicity of psychrophilic alanine racemase by site-directed mutagenesis

A psychrophilic alanine racemase from Bacillus psychrosaccharolyticus has a higher catalytic activity than a thermophilic alanine racemase from Bacillus stearothermophilus even at 60 °C in the presence of pyridoxal 5′-phosphate (PLP), although the thermostability of the former enzyme is lower than that of the latter one [FEMS Microbial. Lett. 192 (2000) 169]. In order to improve the thermostability of the psychrophilic enzyme, two hydrophilic amino acid residues (Glu150 and Arg151) at a surface loop surrounding the active site of the enzyme were substituted with the corresponding residues (Val and Ala) in the B. stearothermophilus alanine racemase. The mutant enzyme (ER150,151VA) showed a higher thermostability, and a markedly lower K_m value for PLP, than the wild type one. In addition, the catalytic activities at low temperatures and kinetic parameters of the two enzymes indicated that the mutant enzyme was more psychrophilic than the wild type one. Thus, the psychrophilic alanine racemase was improved in both psychrophilicity and thermostability by the site-directed mutagenesis. The mutant enzyme may be useful for the production of stereospecifically deuterated NADH and various -amino acids.     

Mechanism-based inactivation of VanX, a D-alanyl-D-alanine dipeptidase necessary for vancomycin resistance

VanX is a zinc-dependent D-Ala-D-Ala amino dipeptidase required for high-level resistance to vancomycin. The enzyme is also able to process dipeptides with bulky C-terminal amino acids [Wu, Z., Wright, G. D., and Walsh, C. T. (1995) Biochemistry 34, 2455-2463]. We took advantage of this observation to design and synthesize the dipeptide-like D-Ala-D-Gly(SPhip-CHF(2))-OH (7) as a potential mechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinone fluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Inhibition of VanX by 7 was time-dependent (K(irr) = 30+/-1 microM; k(inact) = 7.3+/- 0.3 min(-1)) and active site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such as sodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicating that the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivate the dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction that leads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from (19)F NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data are consistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site and the presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D. E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500+/-500 suggest that the inhibitor is likely to react at the entrance of the active site cavity.     

The active site of the junction-resolving enzyme T7 endonuclease I

Endonuclease I is a junction-resolving enzyme encoded by bacteriophage T7, that selectively binds and cleaves four-way DNA junctions. We have recently solved the structure of this dimeric enzyme at atomic resolution, and identified the probable catalytic residues. The putative active site comprises the side-chains of three acidic amino acids (Glu20, Asp55 and Glu65) together with a lysine residue (Lys67), and shares strong similarities with a number of type II restriction enzymes. However, it differs from a typical restriction enzyme as the proposed catalytic residues in both active sites are contributed by both polypeptides of the dimer. Mutagenesis experiments confirm the importance of all the proposed active site residues. We have carried out in vitro complementation experiments using heterodimers formed from mutants in different active site residues, showing that Glu20 is located on a different monomer from the remaining amino acid residues comprising the active site. These experiments confirm that the helix-exchanged architecture of the enzyme creates a mixed active site in solution. Such a composite active site structure should result in unilateral cleavage by the complemented heterodimer; this has been confirmed by the use of a cruciform substrate. Based upon analogy with closely similar restriction enzyme active sites and our mutagenesis experiments, we propose a two-metal ion mechanism for the hydrolytic cleavage of DNA junctions.     

Two conserved threonines collaborate in the Escherichia coli leucyl-tRNA synthetase amino acid editing mechanism

The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have employed an editing mechanism to ensure fidelity in this first step of protein synthesis. The amino acid editing active site for Escherichia coli leucyl-tRNA synthetase resides within the CP1 domain that folds discretely from the main body of the enzyme. A portion of the editing active site is lined with conserved threonines. Previously, we identified one of these threonine residues (Thr(252)) as a critical amino acid specificity factor. On the basis of X-ray crystal structure information, two other nearby threonine residues (Thr(247) and Thr(248)) were hypothesized to interact with the editing substrate near its cleavage site. Single mutations of either of these conserved threonine residues had minimal effects on amino acid editing. However, double mutations that deleted the hydroxyl group from the neighboring threonine residues abolished amino acid editing activity. We propose that these threonine residues, which are also conserved in the homologous isoleucyl-tRNA synthetase and valyl-tRNA synthetase editing active sites, play a central role in amino acid editing. It is possible that they collaborate in stabilizing the transition state.     

Active site specificity of plasmepsin II.

Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.     

Crystal structure of FabG4 from Mycobacterium tuberculosis reveals the importance of C-terminal residues in ketoreductase activity

Rv0242c, also known as FabG4, is a beta-ketoacyl CoA reductase in Mycobacterium tuberculosis. The crystal structure of C-terminal truncated FabG4 is solved at 2.5? resolution which shows the presence of two distinct domains, domain I and II. Domain I partially resembles "flavodoxin type domain" and the domain II is a typical "ketoacyl CoA reductase (KAR) domain". The enzyme exhibits ketoacyl CoA reductase activity by reducing acetoacyl CoA to 3-hydroxyacyl CoA in presence of NADH. Conserved catalytic triad Ser347, Tyr360, and Lys364 constitute the active site residues of the KAR domain. Presence of the Tyr and the Lys residues in the triad in a particular orientation is imperative for effective catalytic mechanism. The importance of loop I and II and the role of the C-terminal residues of KAR domain are highlighted. Comparative structural analyses clearly demonstrate that loop II is stabilized by hydrophobic interaction with C-terminal residues to sustain the orientation of Tyr360. Loop I interacts with loop II via H-bonding network to restrict the active site residue Lys364 in a catalytically favorable orientation.     

Binding modes of inhibitors of ribonuclease T1 as elucidated by analysis of two-dimensional NMR

Aromatic proton and high field shifted methyl proton resonances of RNase T1 complexed with Guo, 2'GMP, 3'GMP, or 5'GMP were assigned to specific amino acid residues by analyses of the two-dimensional NMR spectra in comparison with the crystal structure of the RNase T1-2'GMP complex. These assignments were subsequently correlated to those of free RNase T1 [Hoffmann & Rüterjans (1988) Eur. J. Biochem. 177, 539-560]. The spatial proximities of amino acid residues as elucidated by NOESY spectra were found to be quite similar among free RNase T1 and the inhibitor complexes, showing that large conformational changes did not occur upon complex formation. However, small but appreciable conformational changes were induced, which were reflected by the systematic chemical shift changes of some amino acid residues in the active site. Furthermore, we confirmed that RNase T1 contains two specific binding sites, one for the guanine base and the other for the phosphate moiety. The inhibitors are forced to adapt their conformations to fit the guanine base and the phosphate moiety to each binding site on the enzyme. This is consistent with our previous studies that 2'GMP and 3'GMP take the syn form as a bound conformation, while 5'GMP takes the anti conformation around glycosidic bonds [Inagaki et al. (1985) Biochemistry 24, 1013-1020]. The slow-exchange process between free and bound forms involving Tyr42 and Tyr45 was found to be specific to the recognition of the guanine base.     

Mutations in the alpha8 loop of human APE1 alter binding and cleavage of DNA containing an abasic site

Recent crystallographic studies reveal loops in human AP endonuclease 1 (APE1) that interact with the major and minor grooves of DNA containing apurinic/apyrimidinic (AP) sites. These loops are postulated to stabilize the DNA helix and the flipped out AP residue. The loop alpha8 interacts with the major groove on the 3' side of the AP site. To determine the essentiality of the amino acids that constitute the alpha8 loop, we created a mutant library containing random nucleotides at codons 222-229 that, in wild-type APE1, specify the sequence NPKGNKKN. Upon expression of the library (2 x 10(6) different clones) in Escherichia coli and multiple rounds of selection with the alkylating agent methyl-methane sulfonate (MMS), we obtained approximately 2 x 10(5) active mutants that complemented the MMS sensitivity of AP endonuclease-deficient E. coli. DNA sequencing showed that active mutants tolerated amino acid substitutions at all eight randomized positions. Basic and uncharged polar amino acids together comprised the majority of substitutions, reflecting the positively charged, polar character of the wild-type loop. Asn-222, Asn-226, and Asn-229 exhibited the least mutability, consistent with x-ray data showing that each asparagine contacts a DNA phosphate. Substitutions at residues 226-229, located nearer to the AP site, that reduced basicity or hydrogen bonding potential, increased Km 2- to 6-fold and decreased AP site binding; substitutions at residues 222-225 exhibited lesser effects. This initial mutational analysis of the alpha8 loop supports and extends the conclusion of crystallographic studies that the loop is important for binding of AP.DNA and AP site incision.     

Deletions in the loop surrounding the iron-sulfur cluster of adrenodoxin severely affect the interactions with its native redox partners adrenodoxin reductase and cytochrome P450(scc) (CYP11A1)

The redox active iron-sulfur center of bovine adrenodoxin is coordinated by four cysteine residues in positions 46, 52, 55 and 92 and is covered by a loop containing the residues Glu-47, Gly-48, Thr-49, Leu-50 and Ala-51. In plant-type [2Fe-2S] ferredoxins, the corresponding loop consists of only four amino acids. The loop is positioned at the surface of the proteins and forms a boundary separating the [2Fe-2S] cluster from solvent. In order to analyze the biological function of the five amino acids of the loop in adrenodoxin (Adx) for this electron transfer protein each residue was deleted by site-directed mutagenesis. The resulting five recombinant Adx variants show dramatic differences among each other regarding their spectroscopic characteristics and functional properties. The redox potential is affected differently depending on the position of the conducted deletion. In contrast, all mutations in the protein loop influence the binding to the redox partners adrenodoxin reductase (AdR) and cytochrome P450(scc) (CYP11A1) indicating the importance of this loop for the physiological function of this iron--sulfur protein.     

Effects of ligands on the mobility of an active-site loop in tyrosine hydroxylase as monitored by fluorescence anisotropy

Fluorescence anisotropy has been used to monitor the effect of ligands on a mobile loop over the active site of tyrosine hydroxylase. Phe184 in the center of the loop was mutated to tryptophan, and the three native tryptophan residues were mutated to phenylalanine to form an enzyme with a single tryptophan residue in the mobile loop. The addition of 6-methyl-5-deazatetrahydropterin to the enzyme resulted in a significant increase in the fluorescence anisotropy. The addition of phenylalanine did not result in a significant change in the anisotropy in the presence or absence of the deazapterin. The K(d) value for the deazapterin was unaffected by the presence of phenylalanine. Qualitatively similar results were obtained with apoenzyme, except that the addition of phenylalanine led to a slight decrease in anisotropy. Frequency-domain lifetime measurements showed that the distribution of lifetimes was unaffected by both the amino acid and deazapterin. Frequency-domain anisotropy analyses were consistent with a decrease in the motion of the sole tryptophan in the presence of the deazapterin. This could be modeled as a decrease in the cone angle for the indole ring of about 12 degrees . The data are consistent with a model in which binding of a tetrahydropterin results in a change in the conformation of the surface loop required for proper formation of the amino acid binding site.