HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyzes the transfer of pyrophosphate from ATP to HP (6-hydroxymethyl-7,8-dihydropterin). This first reaction in the folate biosynthetic pathway is a potential target for antimicrobial agents. A Hamiltonian replica exchange method (HREM) molecular dynamics (MD) approach is used, with the goal of improving conformational sampling, whereby multiple copies of the system are run without requiring a large number of System copies. For HPPK, the aim is to improve conformational sampling around the HP binding pocket and thereby find near-closed conformations (similar but not identical to the binding pocket of HP, as defined by the ternary crystal structure). Near-closed conformations may be better targets for the design of species-selective inhibitors. Well-populated, near-closed conformations of Escherichia coli HPPK (EcHPPK) and Yersinia pestis HPPK (YpHPPK) were found with HREM by focusing Oil the interactions involving loops 2 and 3 that are known to be the more flexible regions of HPPK. A small number of systems were found to be sufficient to enlarge the sample space substantially, on the basis of root-mean-square fluctuation measures, relative to the results of a conventional MD simulation. By clustering snapshots on the basis of some of the key residues that form the HP binding pocket, distinct HREM-generated conformations are found. Residue displacements mainly from loop 2 are responsible for the distinct conformers found, relative to the crystal Structure, for both EcHPPK and YpHPPK. In contrast, the conventional MD simulations of EcHPPK and YpHPPK each lead essentially to one cluster, with use of the same Clustering criterion as for the HREM. The shapes of the HREM near-closed binding pockets are qualitatively investigated and found to be different. Some of these conformations are distinguishable between EcHPPK and YpHPPK, indicating that there may be differing species-selective, near-closed conformations suited to HP binding.