Spectrophotometric determination of 3, 4-dihydroxy-2-butanone-4-phosphate synthase activity.
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Base Sequence↗
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Homology of 1-aminocyclopropane-1-carboxylate synthase, 8-amino-7-oxononanoate synthase, 2-amino-6-caprolactam racemase, 2,2-dialkylglycine decarboxylase, glutamate-1-semialdehyde 2,1-aminomutase and isopenicillin-N-epimerase with aminotransferases.
Profile analysis showed the title enzymes to be homologous with the aminotransferases. 1-Aminocyclopropane-1-carboxylate synthase is closely related to subgroup I of aminotransferases which includes aspartate, alanine, histidinol-phosphate, tyrosine and phenylalanine aminotransferase. 2,2-Dialkylglycine decarboxylase, glutamate-1-semialdehyde 2,1-aminomutase and 2-amino-6-caprolactam racemase are most similar to subgroup II which comprises aminotransferases with omega-amino acids as substrates. 8-Amino-7-oxononanoate synthase is closely related to both subgroup I and II, and isopenicillin-N-epimerase to subgroup IV with serine and phosphoserine aminotransferase. Aminotransferases and the title enzymes belong to a regio-specific family of evolutionarily related pyridoxal-5'-phosphate-dependent enzymes.
Acyltransferases↗
Molecular cloning of the human gene encoding lanosterol synthase from a liver cDNA library.
Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] catalyzes the cyclization of (S)-2,3-oxidosqualene to lanosterol in the reaction that forms the sterol nucleus. We report herein the cloning and characterization of the human gene (OSC) encoding lanosterol synthase, a predicted 83 kDa protein of 732 amino acids. The deduced amino acid sequence is 36-40% identical to known yeast and plant homologues and 83% identical to Rattus norvegicus lanosterol synthase. The new gene was shown to encode lanosterol synthase. The yeast lanosterol synthase deficient mutant SMY8 was complemented by the human gene, and a cell-free homogenate of SMY8 expressing the human gene was shown to convert 2,3-oxidosqualene to lanosterol.
Amino Acid Sequence↗
Molecular cloning of a Schizosaccharomyces pombe cDNA encoding lanosterol synthase and investigation of conserved tryptophan residues.
A Schizosaccharomyces pombe cDNA encoding lanosterol synthase was cloned by complementing a Saccharomyces cerevisiae lanosterol synthase mutant. The predicted 83-kDa protein is 54-58% identical to other lanosterol synthases. The previously known lanosterol synthases contain 229 conserved residues, which should encompass the catalytically essential amino acids. This number is decreased dramatically by including the Sc. pombe lanosterol synthase in the analysis; 42 residues are no longer conserved and therefore are catalytically nonessential. We have begun mutagenic studies to identify catalytic residues from the remaining conserved residues. Mutant Sa. cerevisiae lanosterol synthase genes were generated in which phenylalanine was specifically substituted for conserved tryptophan residues. All of the resultant mutant enzymes retained the ability to complement the Sc. cerevisiae lanosterol synthase mutant, suggesting that these conserved tryptophan residues are not catalytically essential.
Amino Acid Sequence↗
The effect of cysteine mutations on recombinant deacetoxycephalosporin C synthase from S. clavuligerus.
Cysteines 100, 155, and 197 of recombinant deacetoxycephalosporin C synthase were mutated to alanine residues. The C100A mutant had properties similar to those of the wild-type enzyme, but mutation of Cys-155 and Cys-197 reduced enzyme activity with penicillin N and penicillin G to different extents.
Amino Acid Substitution↗
The invariant F283 and its strategic position in the hydrophobic cleft of Streptomyces jumonjinensis isopenicillin N synthase active site are functionally important.
Isopenicillin N synthase (IPNS) and related non-haem iron-binding enzymes including deacetoxycephalosporin C synthase (DAOCS) are proposed to have structurally similar active centers. Sequence alignment and computational structural analyses of predicted structures revealed 11 highly conserved hydrophobic amino acid residues in 134 IPNS-related enzymes form a contiguous hydrophobic patch in the IPNS active center, wherein F283 is strategically positioned. The investigation of single and double mutations at F283, adjacent (L284) and proximal sites (N285 and S216) of Streptomyces jumonjinensis IPNS advocate the explicit importance of the phenyl ring at position 283. A similarly placed phenylalanine (F264) in DAOCS was found to be also crucial for its enzyme activity.
Amino Acid Sequence↗
Riboflavin synthesis genes ribE, ribB, ribH, ribA reside in the lux operon of Photobacterium leiognathi.
Nucleotide sequence of the riboflavin synthesis genes ribE, ribB, ribH, ribA (GenBank Accession No. AF364106) resided in the lux operon of Photobacterium leiognathi PL741 has been determined, and the amino acid sequences of riboflavin synthetase (RibE), DHBP synthetase (RibB), lumazine synthetase (RibH), GTP cyclohydrolase II (RibA) encoded by the riboflavin synthesis genes are deduced. Nucleotide sequence reveals that the ribE gene encodes the riboflavin synthetase responsible for converting lumazine to riboflavin, the ribB gene encodes the DHBP synthetase responsible for 3,4-dihydroxyl-2-butanone 4-phosphate synthesis, the ribH gene encodes the lumazine synthetase responsible for lumazine synthesis, and the ribA gene encodes the GTP cyclohydrolase II responsible for lumazine synthesis. Functional analysis illustrates that the specific segments lay behind the ribH and ribA genes might form potential loops Omega(oT) and Omega(TI)--Omega(TII); Omega(oT) is functioned as mRNA stability loop or/and for subregulation by alternative modulation, and Omega(TI)--Omega(TII) could be the transcriptional terminator of the lux operon. The gene order of the ribE, ribB, ribH, ribA genes resided in the lux operon and linked to the lum operon is <--ter*-lumQ-lumP-R&R-luxC-luxD-luxA-luxB-luxN-luxE-luxG-ribE-ribB-ribH-ribA-ter--> (R&R: regulatory region; ter: transcriptional terminator), whereas the R&R is the regulatory region for the lum and the lux operons, and ter and ter* are the transcriptional terminators for the lux and lum operons.
Amino Acid Sequence↗
Mutation of N304 to leucine in Streptomyces clavuligerus deacetoxycephalosporin C synthase creates an enzyme with increased penicillin analogue conversion.
Superimposition of deacetoxycephalosporin C synthase (DAOCS) and isopenicillin N synthase (IPNS) structures revealed that R74, R160, R266 and N304 are strategically located in the catalytic cavity of Streptomyces clavuligerus DAOCS (scDAOCS) and are crucial for orchestrating different substrates. Substitutions at these sites to a hydrophobic leucine residue were expected to stabilize the hydrophobic substrate bound state. Substantial improvements in the biotransformation of penicillin G, ampicillin and amoxicillin to their respective cephalosporin moieties were observed using the N304L mutant scDAOCS. Thus, our results have demonstrated the enhancement of scDAOCS activity via critical computational analysis and site-directed mutagenesis of endogenous ligands.
Amino Acid Substitution↗
Active site mutations of recombinant deacetoxycephalosporin C synthase.
Site-directed mutagenesis of active site residues of deacetoxycephalosporin C synthase active site residues was carried out to investigate their role in catalysis. The following mutations were made and their effects on the conversion of 2-oxoglutarate and the oxidation of penicillin N or G were assessed: M180F, G299N, G300N, Y302S, Y302F/G300A, Y302E, Y302H, and N304A. The Y302S, Y302E, and Y302H mutations reduced 2-oxoglutarate conversions and abolished (<2%) penicillin G oxidation. The Y302F/G300A mutation caused partial uncoupling of penicillin G oxidation from 2-oxoglutarate conversion, but did not uncouple penicillin N oxidation from 2-oxoglutarate conversion. Met-180 is involved in binding 2-oxoglutarate, and the M180F mutation caused uncoupling of 2-oxoglutarate from penicillin oxidation. The N304A mutation apparently enhanced in vitro conversion of penicillin N but had little effect on the oxidation of penicillin G, under standard assay conditions.
Binding Sites↗
PLP and PMP radicals: a new paradigm in coenzyme B6 chemistry.
Enzymes frequently rely on a broad repertoire of cofactors to perform chemically challenging transformations. The B6 coenzymes, composed of pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP), are used by many transaminases, racemases, decarboxylases, and enzymes catalyzing alpha,beta and beta,gamma-eliminations. Despite the variety of reactions catalyzed by B6-dependent enzymes, the mechanism of almost all such enzymes is based on their ability to stabilize high-energy anionic intermediates in their reaction pathways by the pyridinium moiety of PLP/PMP. However, there are two notable exceptions to this model, which are discussed in this article. The first enzyme, lysine 2,3-aminomutase, is a PLP-dependent enzyme that catalyzes the interconversion of L-lysine to L-beta-lysine using a one-electron-based mechanism utilizing a [4Fe-4S] cluster and S-adenosylmethionine. The second enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase, is a PMP-dependent enzyme involved in the formation of 3,6-dideoxysugars in bacteria. This enzyme also contains an iron-sulfur cluster and uses a one-electron based mechanism to catalyze removal of a C-3 hydroxy group from a 4-hexulose. In both cases, the participation of free radicals in the reaction pathway has been established, placing these two B6-dependent enzymes in an exclusive class by themselves.
Catalysis↗
Stimulation of mitogenic pathways through kinase-impaired mutants of the epidermal growth factor receptor.
Two residues have been shown to be critical for the kinase activity of the receptor for epidermal growth factor (EGF): lysine-721, which functions in the binding of ATP by correctly positioning the gamma-phosphate for phosphoryl transfer, and aspartate-813, which functions as the catalytic base of the kinase. Mutation of either of these two residues has been shown to disrupt kinase activity of the receptor. However, studies performed in different laboratories had suggested that while EGF receptors mutated at lysine-721 are unable to stimulate significant increases of [(3)H]thymidine incorporation into DNA in response to EGF treatment, cells expressing EGF receptors mutated at aspartate-813 do stimulate significant incorporation of [(3)H]thymidine into DNA in response to EGF. In the present study, EGF receptors mutated at lysine-721 or aspartate-813 (K721R and D813A, respectively), as well as wild-type EGF receptors, were expressed in the same cellular background, Chinese hamster ovary cells, and side-by-side experiments were performed to investigate possible signaling-related differences. Our results indicate that while there are measurable differences in the abilities of the two mutant receptors to stimulate [(3)H]thymidine incorporation between 20 and 24 h after addition of EGF, these differences cannot be correlated with significant differences in EGF-stimulated tyrosine phosphorylation of mutant EGF receptor and endogenous ErbB2, the extent of receptor internalization, EGF-stimulated ion uptake, stimulation of SHC activity, or receptor association with Grb2. Flow cytometric data suggest that populations of cells expressing either kinase-impaired mutant EGF receptor progress similarly into S phase in response to addition of EGF. These observations suggest that D813A and K721R retain similar ability to stimulate mitogenic signaling events through transactivation of ErbB2 with only subtle temporal differences, and they emphasize the importance of expressing mutant receptors in an identical cellular context to make valid comparisons of functions.
Adaptor Proteins, Signal Transducing↗
Posttranscriptional regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in lens epithelial cells by mevalonate-derived nonsterols.
The ocular lens must continuously synthesize the cholesterol required to support membrane formation for its life-long growth. The roles of transcriptional and posttranscriptional mechanisms in controlling 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) protein levels in cultured lens epithelial cells were examined by measuring the effect of restricting exogenous cholesterol, endogenous cholesterol synthesis and mevalonate derived nonsterols upon HMGR protein and mRNA levels and upon the synthesis and degradation of HMGR protein. Sterols were restricted by culturing in lipoprotein deficient media and blocking 2,3-oxidosqualene cyclase with U18666A. Mevalonate derived nonsterols were additionally restricted by inhibition of HMGR activity with lovastatin. A 4-fold increase in HMGR protein levels due to restricting sterols with U18666A could be explained by comparably increased mRNA levels and enzyme protein synthesis. The very rapid turnover of HMGR protein (T(1/2) approximately 45 min) was unaffected. The additional restriction of mevalonate derived nonsterols increased HMGR protein levels to about 400-fold. A 10-fold slowing in the rate of enzyme degradation coupled with at least a 5-fold increase in mRNA levels likely accounted for this accumulated protein mass. The capacity of the nonsterol regulators to promote enzyme degradation appeared independent of sterols, since mevalonate restored rapid degradation of HMGR protein when 2,3-oxidosqualene cyclase activity was simultaneously blocked. Thus, in cultured lens epithelial cells, sterols appear to exert a modest influence on HMGR protein levels solely by suppressing transcription; whereas, mevalonate derived nonsterols exert major influence mainly by accelerating enzyme protein degradation. We speculate that nonsterol isoprenes might be important for preventing overexpression of cholesterol biosynthesis in the intact lens.
Androstenes↗
Crystallization and preliminary X-ray analysis of wild-type and K272A mutant glutamate 1-semialdehyde aminotransferase from Synechococcus.
Crystals of the pyridoxal-5'-phosphate dependent enzyme glutamate-1-semialdehyde aminotransferase (EC 5.4.3.8) from Synechococcus have been grown from polyethylene glycol solutions. The wild-type enzyme crystallizes in space group P2(1)2(1)2(1), with cell dimensions a = 68.4 A, b = 108.0 A, c = 122.6 A. The inactive mutant in which the cofactor-binding lysine 272 residue is replaced by alanine (K272A) gives monoclinic crystals of space group P2(1) with cell dimensions a = 67.1 A, b = 108.6 A, c = 124.5 A and beta = 115.7 degrees. These crystal forms diffract to 2.4 A and 2.7 A resolution, respectively.
Crystallization↗
Major identity determinants for enzymatic formation of ribothymidine and pseudouridine in the T psi-loop of yeast tRNAs.
Almost all transfer RNA molecules sequenced so far contain two universal modified nucleosides at positions 54 and 55, respectively: ribothymidine (T54) and pseudouridine (psi 55). To identify the tRNA elements recognized by tRNA:m5uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase from the yeast Saccharomyces cerevisiae, a set of 43 yeast tRNA(Asp) mutants were used. Some variants contained point mutations, while the others included progressive reductions in size down to a tRNA minisubstrate consisting of the T psi-loop with only one G.C base-pair as stem (9-mer). All substrates (full-sized tRNA(Asp) and various minihelices) were produced in vitro by T7 transcription and tested using yeast extract (S100) as a source of enzymatic activities and S-adenosyl-L-methionine as a methyl donor. The results indicate that the minimal substrate for enzymatic formation of psi 55 is a stem/loop structure with only four G.C base-pairs in the stem, while a longer stem is required for efficient T54 formation. None of the conserved nucleotides (G53, C56, A58 and C61) and U54 for psi 55 or U55 for T54 formation can be replaced by any of the other three canonical nucleotides. Yeast tRNA:m5uridine-54 methyltransferase additionally requires the presence of a pyrimidine-60 in the loop. Interestingly, in a tRNA(Asp) variant in which the T psi-loop was permuted with the anticodon-loop, the new U32 and U33 residues derived from the T psi-loop were quantitatively converted to T32 and psi 33, respectively. Structural mapping of this variant with ethylnitrosourea confirmed that the intrinsic characteristic structure of the T psi-loop was conserved upon permutation and that the displaced anticodon-loop did not acquire a T psi-loop structure. These results demonstrate that a local conformation rather than the exact location of the U-U sequence within the tRNA architecture is the important identity determinant for recognition by yeast tRNA:m5uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase.
Base Sequence↗
The structure of the membrane protein squalene-hopene cyclase at 2.0 A resolution.
Squalene cyclases catalyze a cationic cyclization cascade, which is homologous to a key step in cholesterol biosynthesis. The structure of the enzyme from Alicyclobacillus acidocaldarius has been determined in a new crystal form at 2.0 A resolution (1 A=0.1 nm) and refined to an R-factor of 15.3 % (Rfree=18.7 %). The structure indicates how the initial protonation and the final deprotonation of squalene occur and how the transient carbocations are stabilized. The pathways of the flexible educt squalene from the membrane interior to the active center cavity and of the rigid fused-ring product hopene in the reverse direction are discussed. The enzyme contains eight so-called QW-sequence repeats that fortify the alpha/alpha-barrels by an intricate interaction network. They are unique to the known triterpene cyclases and are presumed to shield these enzymes against the released enthalpy of the highly exergonic catalyzed reaction. The enzyme is a monotopic membrane protein, the membrane-binding interactions of which are described and compared with those of two prostaglandin-H2 synthase isoenzymes, the only other structurally characterized proteins of this type. In the crystals the membrane-binding regions face each other, suggesting a micelle-type detergent structure between them.
Amino Acid Sequence↗
Studies on the active site of deacetoxycephalosporin C synthase.
The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel filtration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized +/-Fe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A resolution and belonged to the R3 space group (unit cell dimensions: a=b=106.4 A, c=71.2 A; alpha=beta=90 degrees, gamma=120 degrees (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry defined by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 A, three nitrogen or oxygen atoms at 2.11 A and one other light atom at 2.04 A. For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A, another O atom at 2.08 A and one at 2.03 A. Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism.
Binding Sites↗
Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS).
Deacetoxycephalosporin C synthase (DAOCS) is an iron(II) and 2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N to deacetoxycephalosporin C, the committed step in the biosynthesis of cephalosporin antibiotics. The crystal structure of DAOCS revealed that the C terminus of one molecule is inserted into the active site of its neighbor in a cyclical fashion within a trimeric unit. This arrangement has hindered the generation of crystalline enzyme-substrate complexes. Therefore, we constructed a series of DAOCS mutants with modified C termini. Oxidation of 2-oxoglutarate was significantly uncoupled from oxidation of the penicillin substrate in certain truncated mutants. The extent of uncoupling varied with the number of residues deleted and the penicillin substrate used. Crystal structures were determined for the DeltaR306 mutant complexed with iron(II) and 2-oxoglutarate (to 2.10 A) and the DeltaR306A mutant complexed with iron(II), succinate and unhydrated carbon dioxide (to 1.96 A). The latter may mimic a product complex, and supports proposals for a metal-bound CO(2) intermediate during catalysis.
Amino Acid Sequence↗
The B(12)-binding subunit of glutamate mutase from Clostridium tetanomorphum traps the nucleotide moiety of coenzyme B(12).
Glutamate mutase from Clostridium tetanomorphum binds coenzyme B(12) in a base-off/His-on form, in which the nitrogenous ligand of the B(12)-nucleotide function is displaced from cobalt by a conserved histidine. The effect of binding the B(12)-nucleotide moiety to MutS, the B(12)-binding subunit of glutamate mutase, was investigated using NMR spectroscopic methods. Binding of the B(12)-nucleotide to MutS was determined to occur with K(d)=5.6(+/-0.7) mM and to be accompanied by a specific conformational change in the protein. The nucleotide binding cleft of the apo-protein, which is formed by a dynamic segment with propensity for partial alpha-helical conformation (the "nascent" alpha-helix), becomes completely structured upon binding of the B(12)-nucleotide, with formation of helix alpha1. In contrast, the segment containing the conserved residues of the B(12)-binding Asp-x-His-x-x-Gly motif remains highly dynamic in the protein/B(12)-nucleotide complex. From relaxation studies, the time constant tau, which characterizes the time scale for the formation of helix alpha1, was estimated to be about 30 micros (15)N and was the same in both, apo-protein and nucleotide-bound protein. Thus, the binding of the B(12)-nucleotide moiety does not significantly alter the kinetics of helix formation, but only shifts the equilibrium towards the structured fold. These results indicate MutS to be structured in such a way, as to be able to trap the nucleotide segment of the base-off form of coenzyme B(12) and provide, accordingly, the first structural clues as to how the process of B(12)-binding occurs.
Amino Acid Motifs↗