PubMed · 4324064
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V J Perez, T J Cicero, B A Bahn. 1971. Ethanol in brain, as assayed by microfluorometry.. https://pubmed.ncbi.nlm.nih.gov/4324064/
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Pig aldehyde reductase containing the active site mutation tyrosine(50) to phenylalanine has been crystallized in the presence of the cofactor NADP(H) to a resolution of 2.2 A. This structure clearly shows loss of the tyrosine hydroxyl group and no other significant perturbations compared with previously determined structures. The mutant binds cofactor (both oxidized and reduced) more tightly than the wild-type enzyme but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone, as determined by fluorescence titrations. Numerous attempts at preparing a ternary complex with a range of small aldehyde substrates were unsuccessful. This result, in addition to the inability of the mutant protein to bind the inhibitor, provides strong evidence for the proposal that the tyrosine hydroxyl group is essential for substrate binding in addition to catalysis.
A 1.9 A resolution X-ray structure of the apo-form of Corynebacterium 2,5-diketo-d-gluconic acid reductase A (2,5-DKGR A), a member of the aldo-keto reductase superfamily, has been determined by molecular replacement using the NADPH-bound form of the same enzyme as the search model. 2,5-DKGR A catalyzes the NADPH-dependent stereo-specific reduction of 2,5-diketo-d-gluconate (2,5-DKG) to 2-keto-l-gulonate, a precursor in the industrial production of vitamin C. An atomic-resolution structure for the apo-form of the enzyme, in conjunction with our previously reported high-resolution X-ray structure for the holo-enzyme and holo/substrate model, allows a comparative analysis of structural changes that accompany cofactor binding. The results show that regions of the active site undergo coordinated conformational changes of up to 8 A. These conformational changes result in the organization and structural rearrangement of residues associated with substrate binding and catalysis. Thus, NADPH functions not only to provide a hydride ion for catalytic reduction, but is also a critical structural component for formation of a catalytically competent form of DKGR A.
High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase (AOX1) promoter. However, the time taken to reach peak product concentration is usually very long ( approximately 240 h). In this paper, we describe the expression of HBsAg in P. pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose. Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant. However, this was offset by continuous antigen production by the GAP clone. In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure. The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones. The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg. The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen. These particles were sensitive to thiol reagents. We also explored the possibility of secreting the GAP expressed HBsAg in P. pastoris. In-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal under the constitutive GAP promoter resulted in secretion of approximately 20 nm HBsAg particles as evidenced by electron microscopy. However, the levels of secreted HBsAg particles were very low, presumably due to the inherent hydrophobicity of the HBsAg molecule and the consequent propensity for membrane association. Our studies show that secretion is not a good strategy for expression of HBsAg in P. pastoris. The data also suggests that intracellular production of HBsAg under the GAP promoter using multicopy expression cassettes can indeed serve as an effective alternative to the AOX1 promoter. Further, the GAP promoter based system obviates the need to use and extensively monitor methanol during recombinant antigen production. Finally, this constitutive system has the potential for continuous culture wherein several batches of recombinant protein-containing biomass can be harvested from a single initial fermentation.