The phylogeny of C/S1 bZIP transcription factors reveals a shared algal ancestry and the pre-angiosperm translational regulation of S1 transcripts.

Basic leucine zippers (bZIPs) form a large plant transcription factor family. C and S1 bZIP groups can heterodimerize, fulfilling crucial roles in seed development and stress response. S1 sequences also harbor a unique regulatory mechanism, termed Sucrose-Induced Repression of Translation (SIRT). The conservation of both C/S1 bZIP interactions and SIRT remains poorly characterized in non-model species, leaving their evolutionary origin uncertain and limiting crop research. In this work, we explored recently published plant sequencing data to establish a detailed phylogeny of C and S1 bZIPs, investigating their intertwined role in plant evolution, and the origin of SIRT. Our analyses clarified C and S1 bZIP orthology relationships in angiosperms, and identified S1 sequences in gymnosperms. We experimentally showed that the gymnosperm orthologs are regulated by SIRT, tracing back the origin of this unique regulatory mechanism to the ancestor of seed plants. Additionally, we discovered an earlier S ortholog in the charophyte algae Klebsormidium flaccidum, together with a C ortholog. This suggests that C and S groups originated by duplication from a single algal proto-C/S ancestor. Based on our observations, we propose a model wherein the C/S1 bZIP dimer network evolved in seed plants from pre-existing C/S bZIP interactions.

Sugar signals and the control of plant growth and development.

Sugars have a central regulatory function in steering plant growth. This review focuses on information presented in the past 2 years on key players in sugar-mediated plant growth regulation, with emphasis on trehalose 6-phosphate, target of rapamycin kinase, and Snf1-related kinase 1 regulatory systems. The regulation of protein synthesis by sugars is fundamental to plant growth control, and recent advances in our understanding of the regulation of translation by sugars will be discussed.

Role of residue 87 in substrate selectivity and regioselectivity of drug-metabolizing cytochrome P450 CYP102A1 M11.

CYP102A1, originating from Bacillus megaterium, is a highly active enzyme which has attracted much attention because of its potential applicability as a biocatalyst for oxidative reactions. Previously we developed drug-metabolizing mutant CYP102A1 M11 by a combination of site-directed and random mutagenesis. CYP102A1 M11 contains eight mutations, when compared with wild-type CYP102A1, and is able to produce human-relevant metabolites of several pharmaceuticals. In this study, active-site residue 87 of drug-metabolizing mutant CYP102A1 M11 was mutated to all possible natural amino acids to investigate its role in substrate selectivity and regioselectivity. With alkoxyresorufins as substrates, large differences in substrate selectivities and coupling efficiencies were found, dependent on the nature of residue 87. For all combinations of alkoxyresorufins and mutants, extremely fast rates of NADPH oxidation were observed (up to 6,000 min(-1)). However, the coupling efficiencies were extremely low: even for the substrates showing the highest rates of O-dealkylation, coupling efficiencies were lower than 1%. With testosterone as the substrate, all mutants were able to produce three hydroxytestosterone metabolites, although with different activities and with remarkably different product ratios. The results show that the nature of the amino acid at position 87 has a strong effect on activity and regioselectivity in the drug-metabolizing mutant CYP102A1 M11. Because of the wide substrate selectivity of CYP102A1 M11 when compared with wild-type CYP102A1, this panel of mutants will be useful both as biocatalysts for metabolite production and as model proteins for mechanistic studies on the function of P450s in general.

Identification of critical residues in novel drug metabolizing mutants of cytochrome P450 BM3 using random mutagenesis.

Previously, we've described a site-directed triple mutant of cytochrome P450 BM3 (BM3) that is able to convert various drugs (van Vugt-Lussenburg, B. M. A., et al. Biochem. Biophys. Res. Commun. 2006, 346, 810-818). In the present study, random mutagenesis was used to improve the activity of this mutant. With three generations of error-prone PCR, mutants were obtained with 200-fold increased turnover toward drug substrates dextromethorphan and 3,4-methylenedioxymethylamphetamine. The initial activities of these mutants were up to 90-fold higher than that of human P450 2D6. These highly active drug metabolizing enzymes have great potential for biotechnology. Using sequencing analysis, the mutations responsible for the increase in activity were determined. The mutations that had the greatest effects on the activity were F81I, E267V, and particularly L86I, which is not located in the active site. Computer modeling studies were used to rationalize the effects of the mutations. This study shows that random mutagenesis can be used to identify novel critical residues, and to increase our insight into P450s.