A novel NADP(+)-dependent formate dehydrogenase (FDH) from five species within the Burkholderia cepacia complex (BCC) is reported here. These FDHs showed dual coenzyme specificity but preferred NADP(+) over NAD(+), a property which has not been reported before. The distribution of this gene was determined among 46 strains from 10 species of the BCC, and was found to be present in several tested strains of B. cepacia, Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia stabilis and Burkholderia pyrrocinia, but absent in Burkholderia ambifaria, Burkholderia vietnamiensis, Burkholderia dolosa, Burkholderia anthina and Burkholderia ubonensis. The complete coding sequences of FDH genes from these five BCC species consisted of 1161 bp encoding a polypeptide of 386 amino acids. The predicted amino acid sequences showed high sequence identity (91-96%) among the five BCC and 65-70% when compared to those of bacterial NAD(+)-dependent FDHs. The apparent K, of the recombinant FDH from B. stabilis 15516 were 55.5 mM, 0.16 mM and 1.43 mM for formate, NADP(+) and NAD(+), respectively. Interestingly, the NAD(+)-dependent FDHs contained the conserved coenzyme binding sequence Gly(Ala)XGlyXXGlyX(17)Asp, while these five NADP(+)-dependent FDHs possessed GlyXGlyXXGlyX(17)Gln. Gln223Asp mutant can reverse coenzyme preference from NADP to NAD(+). Therefore, Gln223 has an important role on coenzyme specificity toward NADP(+). (C) 2010 Elsevier Inc. All rights reserved.