The DNA sequences of the Thermomonosporafusca genes encoding cellulases E2 and E5 and the N-terminal end of E4 were determined. Each sequence contains an identical 14-bp inverted repeat upstream of the initiation codon. There were no significant homologies between the coding regions of the three genes. The E2 gene is 73% identical to the celA gene from Microbispora bispora, but this was the only homology found with other cellulase genes. E2 belongs to a family of cellulases that includes celA from M. bispora, cenA from Cellulomonasfimi, casA from an alkalophilic Streptomyces strain, and cellobiohydrolase II from Trichoderma reesei. E4 shows 44% identity to an avocado cellulase, while E5 belongs to the Bacillus cellulase family. There were strong similarities between the amino acid sequences of the E2 and E5 cellulose binding domains, and these regions also showed homology with C. fimi and Pseudomonas fluorescens cellulose binding domains.An important step toward understanding the mechanism of action of an enzyme is the determination of its amino acid sequence. In recent years, this usually has been done by determining the DNA sequence of the structural gene that encodes the protein, as DNA sequencing is simpler and more precise than protein sequencing. The sequences of a number of cellulase genes have been determined, and this work has been reviewed by Beguin et al. (1).We have been studying the cellulases of a thermophilic, filamentous soil bacterium, Thermomonospora fusca, and have purified five antigenically distinct cellulases, designated E1 to E5, from the culture supernatant of an extracellularprotease-negative mutant of T. fusca (34). All five enzymes are P-1,4-endoglucanases, but they show considerable variation in their specific activities on several substrates and in their physical properties. The enzymes from T. fusca are heat stable and active over a broad pH range with an optimum centered at pH 6.5. While no complex formation between the cellulases has been seen, enzyme E3 acts synergistically with E2 and E5. Evidence for coordinate regulation (20,22) As part of our study of enzymatic mechanisms of cellulose degradation, we determined the DNA sequences of the structural genes encoding three (E2, E4, E5) of the five purified T. fusca cellulases. Comparisons of the amino acid sequences of these cellulases with each other and with other cellulases yielded information about the similarities and differences among cellulases. Such comparisons may provide insight into the catalytic and regulatory mechanisms of these enzymes. MATERIALS AND METHODSBacterial strains and plasmids. The host strain for all transformations and transfections was Escherichia coli JM101 (rK' mK' supE thi A(/ac-proAB) [F' traD36 proAB lacl"ZAM15]) (36), except for the subcloning of the E4 gene, for which E. coli HB101 (F-hsdS20 [rB-mB-] supE44 * Corresponding author. ara-14 ga/K2 lacYl proA2 rspL20 xyl-5 mtl-l recA13) was used. The cellulase genes were cloned from T. fiusca YX, acquired from Dexter Bellamy, Cornell University (3). T...
Thermomonospora fusca chromosomal DNA was partially digested with EcoRI to obtain 4- to 14-kilobase fragments, which were used to construct a library of recombinant phage by ligation with EcoRI arms of lambda gtWES. lambda B. A recombinant phage coding for xylanase activity which contained a 14-kilobase insert was identified. The xylanase gene was localized to a 2.1-kilobase SalI fragment of the EcoRI insert by subcloning onto pBR322 and derivatives of pBR322 that can also replicate in Streptomyces lividans. The xylanase activity produced by S. lividans transformants was 10- to 20-fold higher than that produced by Escherichia coli transformants but only one-fourth the level produced by induced T. fusca. A 30-kilodalton peptide with activity against both Remazol brilliant blue xylan and xylan was produced in S. lividans transformants that carried the 2.1-kilobase SalI fragment of T. fusca DNA and was not produced by control transformants. T. fusca cultures were found to contain a xylanase of a similar size that was induced by growth on xylan or Solka Floc. Antiserum directed against supernatant proteins isolated from a Solka Floc-grown T. fusca culture inhibited the xylanase activity of S. lividans transformants. The cloned T. fusca xylanase gene was expressed at about the same level in S. lividans grown in minimal medium containing either glucose, cellobiose, or xylan. The xylanase bound to and hydrolyzed insoluble xylan. The cloned xylanase appeared to be the same as the major protein in xylan-induced T. fusca culture supernatants, which also contained at least three additional minor proteins with xylanase activity and having apparent molecular masses of 43, 23, and 20 kilodaltons.
Glandular trichomes of the wild tomato Lycopersicon pennellii Corr. (D'Arcy) secrete large amounts of 2,3,4-tri-O-acylglucoses possessing straight-and branchedchain fatty acids of short to medium chain length (C4-C12).Although previous biosynthetic studies suggested that glucose acylation proceeded via acyl CoA intermediates, repeated attempts to demonstrate isobutyryl-CoA-dependent glucose acylation were unsuccessful. When [14C]isobutyrate is administered to detached L. pennelii leaves, the label is readily molecules. This appears to be the primary mechanism of activation and fatty acid esterification to glucose in L. penneUii trichomes. Cultivated tomato, L. esculentum Mill., also activates free fatty acids to their 1-O-acyl-j3D-glucose derivatives but lacks the acyl transfer mechanism for synthesizing polyacylated sugars.Activation of fatty acids and acylation to form acyl lipids is known to proceed via two pathways. In chloroplasts the activated acyl donor is provided by acyl carrier protein, whereas in the cytoplasm CoA provides the activated acyl group. In both cases activation of the fatty acid is accomplished through a thioester intermediate. In plants, acyl lipids comprise a major fraction of membranes, storage lipids, and the waxy cuticular layer offoliage and roots. Foliar glandular trichomes of the wild tomato Lycopersicon pennellii secrete an unusual class of epicuticular acyl lipids (1). These polar lipids are composed of glucose acylated at positions 2, 3, and 4 with short-chain branched acids (C4 and C5, 60%) and medium-chain length straight and branched fatty acids (C10-C12, 40%). The 2,3,4-tri-O-acylglucoses of L. pennellii are potent insect feeding deterrents (2, 3) and accumulate on the leaf surface at levels as high as 20% of leaf dry weight (4,5 formed as acyl-CoA derivatives via oxidative decarboxylation of a-keto derivatives of branched amino acids (6). These short-chain products act as primers for elongation by a fatty acid synthetase system to produce the C10-C12 medium-chain acyl constituents. Burke et al.(1) noted the similarity of L. pennellii glucolipid acyl substitution to that of triacylglycerols and proposed that the terminal steps of triacylglucose and triacylglycerol biosynthesis could share many similarities. In vivo we observed that acyl precursors were incorporated into triacylglucose with the following efficacy: amino acidoxo-acid >> free acid, suggesting that substrates capable of undergoing oxidative carboxylation and activation as CoA derivatives were more effective substrates. Working with this knowledge and the precedent of cytoplasmic fatty acyl activation through thioester intermediates, we explored the possibility that glucose acylation is acyl-CoA-dependent. However, despite repeated attempts we were unable to demonstrate acyl-CoA-dependent glucose acylation by L. pennellii glandular trichomes. Because we were able to detect palmitoyl-CoA:glycerol-3-phosphate acyltransferase and failed to detect significant acyl-CoA-dependent thioesterase activity in glandu...
Thermomonosporafusca YX grown in the presence of cellulose produces a number of 0-1-4-endoglucanases, some of which bind to microcrystalline cellulose. By using a multicopy plasmid, pUJ702, a gene coding for one of these enzymes (E2) was cloned into Streptomyces lividans and then mobilized into both Escherichia coli and Streptomyces albus. The gene was localized to a 1.6-kilobase PvuII-ClaI segment of the originally cloned 3.0-kilobase SstI fragment of Thermomonospora DNA. The culture supernatants of Streptomyces transformants contain a major endoglucanase that cross-reacts with antibody against Thermomonospora cellulase E2 and has the same molecular weight (43,000) as T. fusca E2. This protein binds quickly and tightly to Avicel, from which it can be eluted with guanidine hydrochloride but not with water. It also binds to filter paper but at a slower rate than to Avicel. Several large proteolytic degradation products of this enzyme generated in vivo lose the ability to bind to Avicel and have higher activity on carboxymethyl cellulose than the native enzyme. Other smaller products bind to Avicel but lack activity. A weak cellobiose-binding site not observed in the native enzyme was present in one of the degradation products. In E. coli, the cloned gene produced a cellulase that also binds tightly to Avicel but appeared to be slightly larger than T. fusca E2. The activity of intact E2 from all organisms can be inactivated by Hg2+ ions. Dithiothreitol protected against Hg2+ inactivation and reactivated both unbound and Avicel-bound Hg2+-inhibited E2, but at different rates. * Corresponding author. simple purification scheme based on the differential binding of E2 and its proteolytic products to Avicel was developed to purify milligram quantities of E2.
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