Probing structural determinants distal to the site of hydrolysis that control substrate specificity of the 20S proteasome

The 20S proteasome is a large multicomponent protease complex. Relatively little is known about the mechanisms that control substrate specificity of its multiple active sites. We present here the crystal structure at 2.95 A resolution of a beta2-selective inhibitor (MB1) bound to the yeast 20S proteasome core particle (CP). This structure is compared to the structure of the CP bound to a general inhibitor (MB2) that covalently modified all three (beta1, beta2, beta5) catalytic subunits. These two inhibitors differ only in their P3 and P4 residues, thereby highlighting binding interactions distal to the active site threonine that control absolute substrate specificity of the complex. Comparisons of the CP-bound structures of MB1, MB2, and the natural products epoxomycin and TMC-95A also provide information regarding general binding modes for several classes of proteasome inhibitors.     

Energetic determinants of oligomeric state specificity in coiled coils

The coiled coil is one of the simplest and best-studied protein structural motifs, consisting of two to five helices wound around each other. Empirical rules have been established on the tendency of different core sequences to form a certain oligomeric state but the physical forces behind this specificity are unclear. In this work, we model four sequences onto the structures of dimeric, trimeric, tetrameric, and pentameric coiled coils. We first examine the ability of an effective energy function (EEF1.1) to discriminate the correct oligomeric state for a given sequence. We find that inclusion of the translational, rotational, and side-chain conformational entropy is necessary for discriminating the native structures from their misassembled counterparts. The decomposition of the effective energy into residue contributions yields theoretical values for the oligomeric propensity of different residue types at different heptad positions. We find that certain calculated residue propensities are general and consistent with existing rules. For example, leucine at d favors dimers, leucine at a favors tetramers or pentamers, and isoleucine at a favors trimers. Other residue propensities are sequence context dependent. For example, glutamine at d favors trimers in one context and pentamers in another. Charged residues at e and g positions usually destabilize higher oligomers due to higher desolvation. Nonpolar residues at these positions confer pentamer specificity when combined with certain residues at positions a and d. Specifically, the pair Leua-Alag' or the inverse was found to stabilize the pentamer. The small energy gap between the native and misfolded counterparts explains why a few mutations at the core sites are sufficient to induce a change in the oligomeric state of these peptides. A large number of possible experiments are suggested by these results.     

Deubiquitinating enzymes: their functions and substrate specificity

Conjugation of one or more molecules of ubiquitin to target proteins can signify one of several fates, including degradation by the 26S proteasome, or trafficking via the secretory or endocytic pathways. Whereas much attention in recent years has focussed on the mechanisms of forming these different ubiquitin conjugates, far less is known about the removal of ubiquitin, which is performed by deubiquitinating enzymes (DUBs). While it has been appreciated for some 10 years that DUBs constitute large gene families in eukaryotes, and known for much longer that ubiquitination is a reversible process, information on the exact role of DUBs has been slow in coming. This review will attempt to summarise results from the last few years that shows that DUBs are an essential regulatory step of both protein degradation by the proteasome, and of other ubiquitin-dependent processes, by virtue of their ability to regulate protein ubiquitination in a target-specific manner.     

Engineered urdamycin glycosyltransferases are broadened and altered in substrate specificity

Combinatorial biosynthesis is a promising technique used to provide modified natural products for drug development. To enzymatically bridge the gap between what is possible in aglycon biosynthesis and sugar derivatization, glycosyltransferases are the tools of choice. To overcome limitations set by their intrinsic specificities, we have genetically engineered the protein regions governing nucleotide sugar and acceptor substrate specificities of two urdamycin deoxysugar glycosyltransferases, UrdGT1b and UrdGT1c. Targeted amino acid exchanges reduced the number of amino acids potentially dictating substrate specificity to ten. Subsequently, a gene library was created such that only codons of these ten amino acids from both parental genes were independently combined. Library members displayed parental and/or a novel specificity, with the latter being responsible for the biosynthesis of urdamycin P that carries a branched saccharide side chain hitherto unknown for urdamycins.     

Structural and dynamical basis of broad substrate specificity, catalytic mechanism, and inhibition of cytochrome P450 3A4

Cytochrome P450 (CYP) 3A4 is responsible for the oxidative degradation of more than 50% of clinically used drugs. By means of molecular dynamics simulations with the newly developed force field parameters for the heme-thiolate group and its dioxygen adduct, we examine the differences in structural and dynamic properties between CYP3A4 in the resting form and its complexes with the substrate progesterone and the inhibitor metyrapone. The results indicate that the broad substrate specificity of CYP3A4 stems from the malleability of a loop (residues 211-218) that resides in the vicinity of the channel connecting the active site and bulk solvent. However, the high-amplitude motion of the flexible loop is found to be damped out upon binding of the inhibitor or the substrate in the active site. In the resting form of CYP3A4, a structural water molecule is bound to the sixth coordination position of the heme iron, stabilizing the octahedral coordination geometry. In addition to the direct coordination of metyrapone to the heme iron, the hydrogen bond interaction between the inhibitor carbonyl group and the side chain of Ser119 also contributes significantly to stabilizing the CYP3A4-metyrapone complex. On the other hand, progesterone is stabilized in the active site by the formation of two hydrogen bonds with Ser119 and Arg106, as well as by the van der Waals interactions with the heme and hydrophobic residues. The structural and dynamic features of the CYP3A4-progesterone complex indicate that the oxidative degradation of progesterone occurs through hydroxylation at the C16 position by the reactive oxygen coordinated to the heme iron.     

Structural specificity in asymmetric charge-transfer complexation of helicenes

The diastereoroeric charge-transfer complexes formed between the two enantiomers of 2,15-dimethoxy[6]helicene and R(?)2-(2,4,5,7-tetranitro- 9-fluorenyloximino)propanoic acid have quite distinct geometries, revealed by NMR.     

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

The traditional method used to investigate the reaction specificity of an enzyme with different substrates is to perform individual kinetic measurements. In this case, a series of varied concentrations are required to study each substrate and a non-regression analysis program is used several times to obtain all the specificity constants for comparison. To avoid the large amount of experimental materials, long analysis time, and redundant data processing procedures involved in the traditional method, we have developed a novel strategy for rapid determination of enzyme substrate specificity using one reaction system containing multiple competing substrates. In this multiplex assay method, the electrospray ionization mass spectrometry (ESI-MS) technique was used for simultaneous quantification of multiple products and a steady-state kinetics model was established for efficient specificity constant calculation. The system investigated was the bacterial sulfotransferase NodH (NodST), which is a host specific nod gene product that catalyzes the sulfate group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to natural Nod factors or synthetic chitooligosaccharides. Herein, the reaction specificity of NodST for four chitooligosaccharide acceptor substrates of different chain length (chitobiose, chitotriose, chitotetraose, and chitopentaose) was determined by both individual kinetic measurements and the new multiplex ESI-MS assay. The results obtained from the two methods were compared and found to be consistent. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiple substrates can be evaluated in one assay.     

Directed evolution and substrate specificity profile of homing endonuclease I-SceI

The laboratory evolution of enzymes with tailor-made DNA cleavage specificities would represent new tools for manipulating genomes and may enhance our understanding of sequence-specific DNA recognition by nucleases. Below we describe the development and successful application of an efficient in vivo positive and negative selection system that applies evolutionary pressure either to favor the cleavage of a desired target sequence or to disfavor the cleavage of nontarget sequences. We also applied a previously described in vitro selection method to reveal the comprehensive substrate specificity profile of the wild-type I-SceI homing endonuclease. Together these tools were used to successfully evolve mutant I-SceI homing endonucleases with altered DNA cleavage specificities. The most highly evolved enzyme cleaves the target mutant DNA sequence with a selectivity that is comparable to wild-type I-SceI's preference for its cognate substrate.     

Structural determinants of inhibitor selectivity in prokaryotic IMP dehydrogenases

The protozoan parasite Cryptosporidium parvum is a major cause of gastrointestinal disease; no effective drug therapy exists to treat this infection. Curiously, C. parvum IMPDH (CpIMPDH) is most closely related to prokaryotic IMPDHs, suggesting that the parasite obtained its IMPDH gene via horizontal transfer. We previously identified inhibitors of CpIMPDH that do not inhibit human IMPDHs. Here, we show that these compounds also inhibit IMPDHs from Helicobacter pylori, Borrelia burgdorferi, and Streptococcus pyogenes, but not from Escherichia coli. Residues Ala165 and Tyr358 comprise a structural motif that defines susceptible enzymes. Importantly, a second-generation CpIMPDH inhibitor has bacteriocidal activity on H. pylori but not E. coli. We propose that CpIMPDH-targeted inhibitors can be developed into a new class of antibiotics that will spare some commensal bacteria.     

Studies on the substrate specificity of Escherichia coli galactokinase

In vitro glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates. Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application. This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK. The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes. [reaction: see text]     

Peptide microarrays for the determination of protease substrate specificity

A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.     

Alterations of lipoxygenase specificity by targeted substrate modification and site-directed mutagenesis

BACKGROUND: Mammalian lipoxygenases (LOXs) are categorised with respect to their positional specificity of arachidonic acid oxygenation. However, the mechanistic basis for this classification is not well understood. To gain a deeper insight into the structural basis of LOX specificity we determined the reaction characteristics of wild-type and mutant mammalian LOX isoforms with native and synthetic fatty acids substrates. RESULTS: The rabbit 15-LOX is capable of catalysing major 12-lipoxygenation when the volume of the substrate-binding pocket is enlarged. These alterations in the positional specificity can be reversed when bulky residues are introduced at the omega end of the substrate. Simultaneous derivatisation of both ends of fatty acids forces a 15-LOX-catalysed 5-lipoxygenation and this reaction involves an inverse head-to-tail substrate orientation. In contrast, for arachidonic acid 5-lipoxygenation by the human 5-LOX the substrate fatty acid may not be inversely aligned. The positional specificity of this isoenzyme may be related to its voluminous substrate-binding pocket. Site-directed mutagenesis, which leads to a reduction of active site volume, converts the 5-LOX to a 15-lipoxygenating enzyme species. CONCLUSIONS: The positional specificity of LOXs is not an invariant enzyme property but depends on the substrate structure and the volume of the substrate-binding pocket. 15-LOX-catalysed 5-lipoxygenation involves an inverse substrate alignment but this may not be the case for 5-LOXs. Thus, both theories for the mechanistic basis of 5-lipoxygenation (straight and inverse substrate orientation) appear to be correct for different LOX isoforms.     

Role and substrate specificity of the Streptomyces coelicolor RedH enzyme in undecylprodiginine biosynthesis

The function of RedH from Streptomyces coelicolor as an enzyme that catalyses the condensation of 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC) and 2-undecylpyrrole to form the natural product undecylprodiginine has been experimentally proven, and the substrate specificity of RedH has been probed in vivo by examining its ability to condense chemically-synthesised MBC analogues with 2-undecylpyrrole to afford undecylprodiginine analogues.     

Changes in substrate specificity of native and recombinant horseradish peroxidase under ionizing radiation

The homogeneous recombinant horseradish peroxidase preparation fromE. coli inclusion bodies exhibits higher specific activity towards ammonium 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) than the native one. The differences in substrate specificity can be assigned to the native enzyme inactivation in the course of metabolic reactions in living plant cells, while the recombinant enzyme reconstructedin vitro completely realizes the original catalytic abilities. Application of the method of radiation-induced inactivation demonstrates the existence of different binding sites for the iodide anion. ABTS, phenol, and guaiacol and allows one to assume a common character of the binding sites of phenol ando-phenylenediamine.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2234–2237, December, 1994.     

Chemoenzymatic synthesis of prodigiosin analogues--exploring the substrate specificity of PigC

Analogues of prodigiosin, a tripyrrolic pigment produced by Serratia species with potent immunosuppressive and anticancer activities, have been produced by feeding synthetic analogues of the normal precursor MBC to mutants of Serratia sp. ATCC 39006 or to engineered strains of Escherichia coli; in this way it has been shown that the prodigiosin synthesising enzyme, PigC, has a relaxed substrate-specificity.