Directed regulation of the promiscuity of pyruvate kinase (PK) on different deoxynucleoside diphosphate (dNDP) substrates is an effective way to improve the process efficiency of biosynthesis of deoxynucleoside triphosphates (dNTP). The rabbit muscle PK was found to be highly specific on adenosine diphosphate (ADP) substrate, and the activity on dNDP substrates were reduced significantly. It was deduced that the bacteria PKs might have better promiscuity on dNDP substrates because of the less demand for fast energy conversion from ADP to ATP by PK. Two bacteria sourced PK genes from Bacillus sp. ATCC 21616 and Zymomonas mobilis ATCC 31821 were cloned and expressed in the E coli strain BL21 (DE3). The results showed that the promiscuity of B. sp. and Z. mobilis PKs on dNDP substrates was improved significantly, which is in agreement with the deduction of bacteria PKs have better substrate promiscuity. The maximum reaction velocities and the Michaelis constants of Z. mobilis and B. sp. PKs on dNDP substrates were within one order of magnitude difference, respectively, comparing to two order of magnitude difference for rabbit muscle PK. The dNDP conversion experiments showed that the process efficiency was improved when the bacteria PKs were used for the dNTP biosynthesis. (C) 2008 Elsevier Inc. All rights reserved.