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A gene encoding l-serine dehydrogenase (l-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciensl-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosal-SerDH, suggesting that P. calidifontisl-SerDH is a novel type of l-SerDH, like Ps. aeruginosal-SerDH. The overexpressed enzyme appears to be the most thermostable l-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards d-serine, in addition to l-serine. Two different crystal structures of P. calidifontisl-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP+/sulfate ion at 1.18 Å and the structure in complex with NADP+/l-tartrate (substrate analog) at 1.57 Å. The fold of the catalytic domain showed similarity with that of Ps. aeruginosal-SerDH. However, the active site structure significantly differed between the two enzymes. Based on the structure of the tartrate, l- and d-serine and 3-hydroxypropionate molecules were modeled into the active site and the substrate binding modes were estimated. A structural comparison suggests that the wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both l- and d-serine enantiomers. This is the first description of the structure of the novel type of l-SerDH with bound NADP+ and substrate analog, and it provides new insight into the substrate binding mechanism of l-SerDH. The results obtained here may be very informative for the creation of l- or d-serine-specific SerDH by protein engineering. [ABSTRACT FROM AUTHOR]

We conducted a comparative analysis of the effects of beryllium fluoride (BeFx) on protein folding mediated by the α- and β-subunit homooligomers (α16mer or β16mer) from the hyperthermophilic archaeum Thermococcus strain KS-1. BeFx inhibited the ATPase activities of both α16mer and β16mer with equal efficiency. This indicated that BeFx replaces the γ-phosphate of chaperonin-bound ATP, thereby forming a stable chaperonin–ADP–BeFx complex. In the presence of ATP and BeFx, both of the two chaperonin subunits mediated green fluorescent protein (GFP) folding. Gel filtration chromatography revealed that the refolded GFP was retained by both chaperonins. Protease digestion and electron microscopic analyses showed that both chaperonin–ADP–BeFx complexes of the two subunits adopted a symmetric closed conformation with the built-in lids of both rings closed and that protein folding took place in their central cavities. These data indicated that basic protein folding mechanisms of α16mer and β16mer are likely similar although there were some apparent differences. While β16mer-mediated GFP refolding in the presence of ATP–BeFx that proceeded more rapidly than in the presence of ATP alone and reached a twofold higher plateau than that achieved with AMP–PNP, the folding mediated by the α16mer that proceeded with much lower yields. A mutant of α16mer, trapα, which traps the unfolded and partially folded substrate protein, did not affect the ATP–BeFx-dependent GFP folding by β16mer but it suppressed that mediated by α16mer to the level of spontaneous folding. These results suggested that β16mer differed from the α16mer in nucleotide binding affinity or the rate of nucleotide hydrolysis. [ABSTRACT FROM AUTHOR]