The hinge-bending motion of the two domains upon closure of the structure, as seen in the Trypanosoma PGK structure, is confirmed. This closed conformation obviously occurs after binding of both substrates and is locked by the Arg62-Asp200 salt bridge. Re-orientations in the conserved active-site loop region around Thr374 also bring both domains into direct contact in the core region of the former inter-domain cleft, to form the complete catalytic site. Comparison of extremely thermostable TmPGK with less thermostable homologues reveals that its increased rigidity is achieved by a raised number of intramolecular interactions, such as an increased number of ion pairs and additional stabilization of alpha helix and loop regions. The covalent fusion with triosephosphate isomerase might represent an additional stabilization strategy.

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The gene for a l(+)‐lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was cloned by complementation of an Escherichia coli pfl. ldh mutant. The gene is part of a 4.5 kb SauIIIA fragment obtained by partial digestion of the Thermotoga genome. The DNA fragment was physically mapped and the putative Shine‐Dalgarno sequence within the non‐coding region determined. The gene contains 960 bp, including the stop codon, corresponding to 319 amino acids/subunit of the homotetrameric enzyme. Part of the amino acid sequence was confirmed by Edman degradation of peptides obtained from nanomolar quantities of the purified enzyme by tryptic digestion. A comparison of the amino acid sequenc̀e with those of known prokaryotic l‐lactate dehydrogenases reveals a high similarity, especially with the enzyme from thermophilic sources, where up to 48% identity is found. The gene was expressed as an active enzyme in a heterologous host.