Do enzymes obey the Baldwin rules? A mechanistic imperative in enzymatic cyclization reactions.
It is commonly assumed that enzymes have evolved to abide by the same energetic and stereoelectronic principles that govern reactions in solution. The principles formulated for organic ring-closure reactions can be used to develop a hypothesis for analysis of enzyme-catalyzed cyclization reactions. (+info)
Purification and characterization of 2-deoxy-scyllo-inosose synthase derived from Bacillus circulans. A crucial carbocyclization enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics.
The biosynthesis of 2-deoxystreptamine, the central aglycon of a major group of clinically important aminoglycoside antibiotics, commences with the initial carbocycle formation step from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. This crucial step is known to be catalyzed by 2-deoxy-scyllo-inosose synthase, which has not yet been characterized so far. Reported in this paper is the first purification of 2-deoxy-scyllo-inosose synthase from butirosin-producing Bacillus circulans SANK 72073 to electrophoretic homogeneity. The enzyme was isolated as a heterodimeric protein comprising from a 23 kDa- and a 42 kDa polypeptide chains. The Km of the enzyme for D-glucose-6-phosphate was estimated to be 9.0 x 10(-4) M and that for NAD+ 1.7 x 10(-4) M, kcat for D-glucose-6-phosphate being 7.3 x 10(-2) s(-1). The presence of Co2+ was essential for the enzyme activity, but Zn2+ was totally inhibitory. While the reaction mechanisms are quite similar, 2-deoxy-scyllo-inosose synthase appears to be distinct from dehydroquinate synthase in the shikimate pathway, with respect to the quaternary structure, metal ion requirement, and the kinetic parameters. (+info)
Elucidating the mechanism of chain termination switching in the picromycin/methymycin polyketide synthase.
BACKGROUND: A single modular polyketide synthase (PKS) gene cluster is responsible for production of both the 14-membered macrolide antibiotic picromycin and the 12-membered macrolide antibiotic methymycin in Streptomyces venezuelae. Building on the success of the heterologous expression system engineered using the erythromycin PKS, we have constructed an analogous system for the picromycin/methymycin PKS. Through heterologous expression and construction of a hybrid PKS, we have examined the contributions that the PKS, its internal thioesterase domain (pikTE) and the Pik TEII thioesterase domain make in termination and cyclization of the two polyketide intermediates. RESULTS: The picromycin/methymycin PKS genes were functionally expressed in the heterologous host Streptomyces lividans, resulting in production of both narbonolide and 10-deoxymethynolide (the precursors of picromycin and methymycin, respectively). Co-expression with the Pik TEII thioesterase led to increased production levels, but did not change the ratio of the two compounds produced, leaving the function of this protein largely unknown. Fusion of the PKS thioesterase domain (pikTE) to 6-deoxyerythronolide B synthase (DEBS) resulted in formation of only 14-membered macrolactones. CONCLUSIONS: These experiments demonstrate that the PKS alone is capable of catalyzing the synthesis of both 14- and 12-membered macrolactones and favor a model by which different macrolactone rings result from a combination of the arrangement between the module 5 and module 6 subunits in the picromycin PKS complex and the selectivity of the pikTE domain. (+info)
Heterologous expression, purification, reconstitution and kinetic analysis of an extended type II polyketide synthase.
BACKGROUND: Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC). RESULTS: The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate. CONCLUSIONS: Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems. (+info)
Protein aging hypothesis of Alzheimer disease.
Alzheimer disease (AD), the most common form of aging-related neurodegenerative disorders, is associated with formation of fibrillar deposits of amyloid beta-protein (Abeta). While the direct involvement of Abeta in AD has been well documented, the relations between Abeta production, amyloid formation, and neurodegeneration remain unknown. We propose that AD is initiated by a protein aging-related structural transformation in soluble Abeta. We hypothesize that spontaneous chemical modification of aspartyl residues in Abeta to transient succinimide induces a non-native conformation in a fraction of soluble Abeta, rendering it amyloidogenic and neurotoxic. Conformationally altered Abeta is characterized by increased stability in solution and the presence of a non-native beta-turn that determines folding of Abeta in solution and the structure of Abeta subunits incorporated into amyloid fibrils. While the soluble 'non-native' Abeta is both the factor triggering the neurodegenerative cascade and the precursor of amyloid plaques, these two events result from interaction of Abeta with different sets of cellular components and need not coincide in space and time. Extensive literature data and experimental evidence are provided in support of this hypothesis. (+info)
Site-directed mutagenesis of squalene-hopene cyclase: altered substrate specificity and product distribution.
BACKGROUND: Two regions of squalene-hopene cyclase (SHC) were examined to define roles for motifs posited to be responsible for initiation and termination of the enzyme-catalyzed polyolefinic cyclizations. Specifically, we first examined the triple mutant of the DDTAVV motif, a region deeply buried in the catalytic cavity and thought to be responsible for the initiation of squalene cyclization. Next, four mutants were prepared for Glu45, a residue close to the substrate entrance channel proposed to be involved in the termination of the cyclization of squalene. RESULTS: The DDTAVV motif in SHC was changed to DCTAEA, the corresponding conserved region of eukaryotic oxidosqualene cyclase (OSC), by the triple mutation of D377C/V380E/V381A; selected single mutants were also examined. The triple mutant showed no detectable cyclization of squalene, but effectively cyclized 2,3-oxidosqualene to give mono- and pentacyclic triterpene products. Of the Glu45 mutants, E45A and E45D showed reduced activity, E45Q showed slightly increased activity, and E45K was inactive. A normal yield of pentacyclic products was produced, but the ratio of hopene 2 to hopanol 3 was significantly changed in the less active mutants. CONCLUSIONS: Initiation and substrate selectivity may be determined by the interaction of the DDTAVV motif with the isopropylidene of squalene (for SHC) and of the DCTAEA motif with the epoxide of oxidosqualene (for OSC). This is the first report of a substrate switch determined by a central catalytic motif in a triterpenoid cyclase. At the termination of cyclization, the product ratio may be largely controlled by Glu45 at the entrance channel to the active site. (+info)
Aureusidin synthase: a polyphenol oxidase homolog responsible for flower coloration.
Aurones are plant flavonoids that provide yellow color to the flowers of some popular ornamental plants, such as snapdragon and cosmos. In this study, we have identified an enzyme responsible for the synthesis of aurone from chalcones in the yellow snapdragon flower. The enzyme (aureusidin synthase) is a 39-kilodalton, copper-containing glycoprotein catalyzing the hydroxylation and/or oxidative cyclization of the precursor chalcones, 2',4',6',4-tetrahydroxychalcone and 2',4',6',3,4-pentahydroxychalcone. The complementary DNA encoding aureusidin synthase is expressed in the petals of aurone-containing varieties. DNA sequence analysis revealed that aureusidin synthase belongs to the plant polyphenol oxidase family, providing an unequivocal example of the function of the polyphenol oxidase homolog in plants, i.e., flower coloration. (+info)
Intramolecular proton transfer in the cyclization of geranylgeranyl diphosphate to the taxadiene precursor of taxol catalyzed by recombinant taxadiene synthase.
BACKGROUND: The committed step in the biosynthesis of the anticancer drug taxol in yew (Taxus) species is the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The enzyme taxadiene synthase catalyzes this complex olefin cation cyclization cascade involving the formation of three rings and three stereogenic centers. RESULTS: Recombinant taxadiene synthase was incubated with specifically deuterated substrates, and the mechanism of cyclization was probed using MS and NMR analyses of the products to define the crucial hydrogen migration and terminating deprotonation steps. The electrophilic cyclization involves the ionization of the diphosphate with closure of the A-ring, followed by a unique intramolecular transfer of the C11 proton to the re-face of C7 to promote closure of the B/C-ring juncture, and cascade termination by proton elimination from the beta-face of C5. CONCLUSIONS: These findings provide insight into the molecular architecture of the first dedicated step of taxol biosynthesis that creates the taxane carbon skeleton, and they have broad implications for the general mechanistic capability of the large family of terpenoid cyclization enzymes. (+info)