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S-adenosylhomocysteine (SAH) is an effective sedative, a good sleep modulator, and a new anticonvulsant. SAH can be synthesized from adenosine and homocysteine by using microbial S-adenosylhomocysteine hydrolase (SAHase). The extremely thermostable SAHase and lactate dehydrogenase (LDH) from Thermotoga maritima were successfully overexpressed in Escherichia coli, and purified by heat treatments. The SAHase exhibited the highest activity at 85°C and pH 8.0 with a specific activity of 6.2U/mg when NAD concentration was 1mM. However, optimal SAHase reaction conditions shifted to 100°C and pH 11.2, and its specific activity increased to 36.8U/mg after NAD concentration was raised to 8mM. Biosynthesis of SAH at 85°C largely increased the adenosine solubility which was a limiting factor for improving the titer of product. At 85°C and pH 8.0, 24μmol of SAH was obtained when 0.5mg of SAHase was applied to a 10ml reaction mixture. The SAH production was further increased to 153μmol by adding LDH and pyruvate into the reaction mixture for NAD regeneration. Therefore, extremely thermostable enzymes SAHase and LDH from T. maritima form an efficient NAD consumption and regeneration system for SAH biosynthesis. This method has great potential for industrial-scale enzymatic production of SAH. [ABSTRACT FROM AUTHOR]

S-Adenosylhomocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosylhomocysteine into adenosine and homocysteine. The SAHH from Thermotoga maritima ( TmSAHH) was expressed in Escherichia coli and the recombinant protein was purified and crystallized. TmSAHH crystals belonging to space group C2, with unit-cell parameters a = 106.3, b = 112.0, c = 164.9 Å, β = 103.5°, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.85 Å resolution. Initial phase determination by molecular replacement clearly indicated that the crystal contains one homotetramer per asymmetric unit. Further refinement of the crystal structure is in progress. [ABSTRACT FROM AUTHOR]

S-adenosylhomocysteine (SAH) hydrolase catalyzes the reversible hydrolysis of SAH into adenosine and homocysteine by using NAD + as a cofactor. The enzyme from Thermotoga maritima (tmSAHH) has great potentials in industrial applications because of its hyperthermophilic properties. Here, two crystal structures of tmSAHH in complex with NAD + show both open and closed conformations despite the absence of bound substrate. Each subunit of the tetrameric enzyme is composed of three domains, namely the catalytic domain, the NAD + -binding domain and the C-terminal domain. The NAD + binding mode is clearly observed and a substrate analogue can also be modeled into the active site, where two cysteine residues in mesophilic enzymes are replaced by serine and threonine in tmSAHH. Notably, the C-terminal domain of tmSAHH lacks the second loop region of mesophilic SAHH, which is important in NAD + binding, and thus exposes the bound cofactor to the solvent. The difference explains the higher NAD + requirement of tmSAHH because of the reduced affinity. Furthermore, the feature of missing loop is consistently observed in thermophilic bacterial and archaeal SAHHs, and may be related to their thermostability. [ABSTRACT FROM AUTHOR]