RESUMEN
The d-2-hydroxyacid dehydrogenase (2HADH) family illustrates a complex evolutionary history with multiple lateral gene transfers and gene duplications and losses. As a result, the exact functional annotation of individual members can be extrapolated to a very limited extent. Here, we revise the previous simplified view on the classification of the 2HADH family; specifically, we show that the previously delineated glyoxylate/hydroxypyruvate reductase (GHPR) subfamily consists of two evolutionary separated GHRA and GHRB subfamilies. We compare two representatives of these subfamilies from Sinorhizobium meliloti (SmGhrA and SmGhrB), employing a combination of biochemical, structural, and bioinformatics approaches. Our kinetic results show that both enzymes reduce several 2-ketocarboxylic acids with overlapping, but not equivalent, substrate preferences. SmGhrA and SmGhrB show highest activity with glyoxylate and hydroxypyruvate, respectively; in addition, only SmGhrB reduces 2-keto-d-gluconate, and only SmGhrA reduces pyruvate (with low efficiency). We present nine crystal structures of both enzymes in apo forms and in complexes with cofactors and substrates/substrate analogues. In particular, we determined a crystal structure of SmGhrB with 2-keto-d-gluconate, which is the biggest substrate cocrystallized with a 2HADH member. The structures reveal significant differences between SmGhrA and SmGhrB, both in the overall structure and within the substrate-binding pocket, offering insight into the molecular basis for the observed substrate preferences and subfamily differences. In addition, we provide an overview of all GHRA and GHRB structures complexed with a ligand in the active site.
Asunto(s)
Oxidorreductasas de Alcohol/química , Aldehído Oxidorreductasas/química , Proteínas Bacterianas/química , Hidroxipiruvato Reductasa/química , Sinorhizobium meliloti/enzimología , Oxidorreductasas de Alcohol/clasificación , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Aldehído Oxidorreductasas/clasificación , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Hidroxipiruvato Reductasa/clasificación , Hidroxipiruvato Reductasa/genética , Hidroxipiruvato Reductasa/metabolismo , Cinética , Modelos Moleculares , Filogenia , Conformación Proteica , Sinorhizobium meliloti/química , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Especificidad por SustratoRESUMEN
The misidentification of a protein sample, or contamination of a sample with the wrong protein, may be a potential reason for the non-reproducibility of experiments. This problem may occur in the process of heterologous overexpression and purification of recombinant proteins, as well as purification of proteins from natural sources. If the contaminated or misidentified sample is used for crystallization, in many cases the problem may not be detected until structures are determined. In the case of functional studies, the problem may not be detected for years. Here several procedures that can be successfully used for the identification of crystallized protein contaminants, including: (i) a lattice parameter search against known structures, (ii) sequence or fold identification from partially built models, and (iii) molecular replacement with common contaminants as search templates have been presented. A list of common contaminant structures to be used as alternative search models was provided. These methods were used to identify four cases of purification and crystallization artifacts. This report provides troubleshooting pointers for researchers facing difficulties in phasing or model building.