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Identification and Characterization of a Second Butyrate Kinase from Clostridium Acetobutylicum ATCC 824

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Identification and Characterization of a Second Butyrate Kinase from Clostridium Acetobutylicum ATCC 824 J. Mol. Microbiol. Biotechnol. (2000) 2(1): 33-38. A Second ButyrateFermentation Kinase from C. acetobutylicumSymposium 33 JMMB Research Article Identification and Characterization of a Second Butyrate Kinase from Clostridium acetobutylicum ATCC 824 Ke-xue Huang, Shouqin Huang, Frederick B. Rudolph*, C. beijerinckii NCIMB 8052 (Oultram et al., 1993) also George N. Bennett clusters with the corresponding ptb gene in a similar arrangement. In addition, butyrate kinase activity has been Department of Biochemistry and Cell Biology, detected in crude extracts of other clostridia, such as Rice University, 6100 Main Street, Houston, Clostridium tetanomorphum and Clostridium pasteurianum TX 77005-1892, USA (Twarog and Wolfe, 1963). In previous work, disruption of the buk gene in Clostridium acetobutylicum ATCC 824 significantly reduced Abstract butyrate kinase activity but did not completely eliminate enzyme activity or butyrate formation (Green et al., 1996). A gene encoding a new butyrate kinase isozyme (BKII) This result prompted us to search for another putative gene was identified from the C. acetobutylicum ATCC 824 encoding for butyrate kinase. Here we report the DNA database. The enzyme was expressed in identification and characterization of a second butyrate Escherichia coli, purified, and characterized. The kinase from C. acetobutylicum ATCC 824. purified enzyme exhibited a subunit molecular mass of 43 kDa by SDS-PAGE, and a native molecular mass Results and Discussion of 80 kDa by gel filtration suggesting it functions as a dimer. In the butyryl phosphate-forming direction the Sequence Analysis optimal pH of BKII was 8.5. The enzyme had a Km of The newly available C. acetobutylicum DNA sequence 5 0.62 M and a turn over rate of 2.2 × 10 /sec (Vmax of 165 database (http://www.ncbi.nlm.nih.gov/BLAST/ units/mg). The presence of a mRNA encoding the BKII unfinishedgenome.html) made possible the identification was demonstrated using a reverse transcription PCR of an additional gene putatively encoding a butyrate kinase reaction. The expression of the BKII in Clostridium activity. We performed a blast analysis on the C. acetobutylicum ATCC 824 was further examined by acetobutylicum DNA sequence database and found an Western blot analysis using a polyclonal antibody open reading frame that showed high homology with the prepared against recombinant BKII. previously cloned butyrate kinase from the same strain (Cary et al., 1998) (similarity 79%, identity 63%), the Introduction butyrate kinase from C. beijerinckii NCIMB 8502 (Oultram et al., 1993) (similarity 78%, identity 61%) and Bacillus Clostridium acetobutylicum has a long history of industrial subtilis branched chain fatty acid kinase (accession number interest because of its biphasic fermentation of sugars to P54532) (similarity 75%, identity 54%) (Figure 1). The produce acetic and butyric acids in the acidogenic phase, previously cloned butyrate kinase gene clusters with PTB, and then acetone, butanol and ethanol in the solvent phase which is an essential gene for biosynthesis of butyrate. (Jones and Woods, 1989). The recent availability of the This newly identified butyrate kinase gene does not cluster genomic sequence for C. acetobutylicum ATCC 824 has with any other identified acid- or solvent-related genes. allowed a more complete analysis of major metabolic enzymes in the organism. Two enzymes, Expression and Purification of BKII phosphotransbutyrylase (PTB) (EC 2.3.1.8) (Valentine and In an initial attempt to express the butyrate kinase gene in Wolf, 1960) and butyrate kinase (BK) (EC 2.7.2.7) E. coli, the full-length butyrate kinase gene was amplified (Hartmanis, 1987) play a key role in the production of by PCR from C. acetobutylicum DNA. A NdeI and XhoI butyrate and in the energy metabolism of the organism fragment from the initial plasmid clone, pCRBK, was (Valentine and Wolf, 1960). BK also catalyzes the reversible inserted into the commonly used expression plasmid, formation of butyryl phosphate from butyrate with pET28a, which carries a T7 promoter. Significant quantities dephosphorylation of ATP, which is an important step in of butyrate kinase were detected in extracts of E. coli utilization of butyrate to form butanol during the BL21(DE3)/pET28BK (data not shown). Unfortunately, the solventogenic phase of fermentation. We have previously expressed protein formed insoluble inclusion bodies even cloned the buk gene encoding butyrate kinase (BKI) (Cary when the concentration of IPTG and temperature of et al., 1988), that clusters with the gene expressing PTB. induction and the host strain were varied. To overcome The functional cloning of this operon from C. acetobutylicum this problem, we turned to the expression vector ATCC 824 was accomplished by complementing the E. pET32a(+),also carrying a T7 promoter and producing a coli atoD32 mutant LJ32 which lacks butyrate kinase activity thioredoxin fusion protein, which enhances the solubility (Jenkins and Nunn, 1987). The buk gene identified from of the expressed protein in E. coli (Lunn et al., 1984; Lavallie et al., 1993; Huang et al., 1998a; Huang et al., 1998b). A histidine tag is also present to allow simple purification. Received September 15, 1999; revised November 17, 1999; accepted The constructed plasmid pET32BK was transformed into November 20, 1999. *For correspondence. Email [email protected]; E. coli BL21(DE3). IPTG induction of the E. coli BL21(DE3)/ Tel. 713-348-4015; Fax. 713-348-5154. pET32BK resulted exclusively in inclusion body formation © 2000 Horizon Scientific Press Further Reading Caister Academic Press is a leading academic publisher of advanced texts in microbiology, molecular biology and medical research. 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The amino acid sequence from C. acetobutylicum ATCC 824 BKI (Accession number L14744), C. beijerinckii NCIMB 8052 butyrate kinase (Accession number L04468); B. subtilis putative branched-chain-fatty-acid kinase (Accession number P54532) are aligned. Positions with 100% identical amino acids are shaded and boxed. A
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