The exocellulase E3 gene was cloned on a 7.1 kb NotI fragment from Thermomonospora fusca genomic DNA into Escherichia coli and expressed in Streptomyces lividans. The E3 gene was sequenced and encoded a 596 residue peptide. The molecular masses of the native and cloned E3s were determined by mass spectrometry, and the value for E. coli E3, 59,797 Da, agreed well with that predicted from the DNA sequence, 59,646 Da. The value of 61,200 Da for T. fusca E3 is consistent with E3 being a glycoprotein. E3 is thermostable, retaining full activity after 16 h at 55 degrees C. It also has a broad pH optimum around 7-8, retaining 90% of its maximal activity between pH 6 and 10. The cloned E3s were identical to the native enzyme in their activity, cellulose binding, and thermostability. Papain digestion produced a 45.7 kDa catalytic domain with 77% of the native activity on amorphous cellulose and 33% on crystalline cellulose. E3 belongs to cellulase family B and retains the residues that have been identified to be crucial for catalytic activity in Trichoderma reesei cellobiohydrolase II and T. fusca E2. The E3 gene contains a 14 bp inverted repeat regulatory sequence 212 bp before the translational start codon instead of the 30-70 bp found for the other T. fusca cellulase genes. An additional copy of this sequence with one base changed is 314 bp before the translational start codon. The transcriptional start site of the E3 gene was shown to be between these two inverted repeats.

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...

Two genes encoding cellulases El and E4 from Thermomonosporafusca have been cloned in Escherichia coli, and their DNA sequences have been determined. Both genes were introduced into Streptomyces lividans, and the enzymes were purified from the culture supernatants of transformants. El and E4 were expressed 18and 4-fold higher, respectively, in S. lividans than in E. coli. Thin-layer chromatography of digestion products showed that El digests cellotriose, cellotetraose, and cellopentaose to cellobiose and a trace of glucose. E4 is poor at degrading cellotriose and cleaves cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. It readily cleaves cellotetraose to cellobiose. El shows 59% identity to Cellulomonas fimi CenC in a 689-amino-acid overlap, and E4 shows 80%o identity to the N terminus of C. fimi CenB in a 441-amino-acid overlap; all of these proteins are members of cellulase family E. Alignment of the amino acid sequences of Clostridium thermocellum celD, El, E4, and four other members of family E demonstrates a clear relationship between their catalytic domains, although there is as little as 25% identity between some of them. Residues in celD that have been identified by site-directed mutagenesis and chemical modification to be important for catalytic activity are conserved in all seven proteins. The catalytic domains of El and E4 are not similar to those of T. fusca E2 or E5, but all four enzymes share similar cellulose-binding domains and have the same 14-bp inverted repeat upstream of their initiation codons. This sequence has been identified previously as the binding

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that forms the SNARE complex with VAMP/synaptobrevin and SNAP-25. Identifying proteins that modulate SNARE complex formation is critical for understanding the molecular mechanisms underlying neurotransmitter release and its modulation. We have cloned and characterized a protein called syntaphilin that is selectively expressed in brain. Syntaphilin competes with SNAP-25 for binding to syntaxin-1 and inhibits SNARE complex formation by absorbing free syntaxin-1. Transient overexpression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls free syntaxin-1 availability for the assembly of the SNARE complex, and thereby regulates synaptic vesicle exocytosis.