TY - JOUR T1 - The tunable intrahexamer heteroassembly mechanism of pseudoenzyme/enzyme pair, PDX1.2/PDX1.3, with its molecular impact on vitamin B<sub>6</sub> regulation JF - bioRxiv DO - 10.1101/717082 SP - 717082 AU - Irina Novikova AU - Mowei Zhou AU - Jared Shaw AU - Marcelina Parra AU - Hanjo Hellmann AU - James E. Evans Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/07/28/717082.abstract N2 - Plants boost the expression of pseudo-enzyme PDX1.2 under heat stress and during embryonic development. PDX1.2 positively regulates vitamin B6 production by hetero-association with its active catalytic homologs such as PDX1.1 and PDX1.3. These heterologous interactions were found challenging to understand. For instance, the crystals of PDX1.2-PDX1.3 heterocomplexes were found to be statistically disordered and individual proteins could not be assigned. Using a combination of biochemical and structural tools, we find that the key to this phenomenon is the nature of PDX1.2 hetero-assembly with its catalytic counterparts. Using a cell-free protein synthesis approach, we were able to set up a precise control of co-expression where we systematically varied the ratios of co-produced proteins by tuning the ratios of input DNA. These were further analyzed by Native Mass Spectrometry, which elucidated that 6-8 hetero-complex species of dodecamers of variable stoichiometry are produced for each co-expression condition tested. This is in contrast to previous hypothesis of stacked inter-hexamer assembly mechanism. As proposed previously, our high-resolution Cryo-EM structure of pseudo-enzyme PDX1.2 closely mimics the fold of PDX1.3 and maintains all necessary protein-protein interactions between subunits. In PDX1.2, the altered catalytic site P1 appears perturbed in concordance with its lack of activity, while the P2 site appears largely unchanged. The most surprising finding is that we observe a complete switch in the surface electrostatics for PDX1.2. Based on the activity assays and its structure, we hypothesize that the change in electrostatic would have a significant impact on the neighboring P2 site of the PDX1.3 and influence the turnover efficiency at that site. These data suggest that pseudo-enzyme PDX1.2 rather acts as an electrostatic tuning module, that, in combination with its hetero-assembly mechanism based on random incorporation, imposes a perfect regulatory control of such important process. ER -