The Clostridium acetobutylicum genome encodes two butyrate kinases. The gene for butyrate kinase I (BKI) is adjacent to the gene for phosphotransbutyrylase and this operon serves a major role in butyrate formation. A second butyrate kinase gene encodes an enzyme (BKII) that exhibits a much higher Km for butyrate and has a more rigid specificity than BKI. We used site specific mutagenesis and protein structural tools to explore the basis of the unique properties of BKII. Several BKII mutants were created and the enzymatic properties were compared considering structural features of other kinase family members. Alignment of the amino acid sequences for the acetate, propionate, and butyrate kinases from both Archaea and Bacteria domains revealed absolute conservation at two critical arginine and two critical histidine residues. High similarity was observed at two glutamate residues. Replacements of residues H155, H183, R215, and E334 resulted in undetectable kinase activity suggesting that these residues are essential. A polar or positively charged residue is required in position 332 and a positively charged residue is required at position 150. The amino acid sequences of both BKI and BKII were homology modeled with the three-dimensional structure of acetate kinase from Methanosarcina thermophila. This is the first structural description of an n-butyrate kinase. (C) 2009 Elsevier Inc. All rights reserved.