Dataset Information

A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY.

ABSTRACT: Light-oxygen-voltage (LOV) domain-containing proteins function as small light-activated modules capable of imparting blue light control of biological processes. Their small modular nature has made them model proteins for allosteric signal transduction and optogenetic devices. Despite intense research, key aspects of their signal transduction mechanisms and photochemistry remain poorly understood. In particular, ordered water has been identified as a possible key mediator of photocycle kinetics, despite the lack of ordered water in the LOV active site. Herein, we use recent crystal structures of a fungal LOV protein ENVOY to interrogate the role of Thr(101) in recruiting water to the flavin active site where it can function as an intrinsic base to accelerate photocycle kinetics. Kinetic and molecular dynamic simulations confirm a role in solvent recruitment to the active site and identify structural changes that correlate with solvent recruitment. In vivo analysis of T101I indicates a direct role of the Thr(101) position in mediating adaptation to osmotic stress, thereby verifying biological relevance of ordered water in LOV signaling. The combined studies identify position 101 as a mediator of both allostery and photocycle catalysis that can impact organism physiology.

SUBMITTER: Lokhandwala J

PROVIDER: S-EPMC4938200 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY.

Lokhandwala Jameela J Silverman Y de la Vega Rafael I RI Hopkins Hilary C HC Britton Collin W CW Rodriguez-Iglesias Aroa A Bogomolni Roberto R Schmoll Monika M Zoltowski Brian D BD

The Journal of biological chemistry 20160516 28

Light-oxygen-voltage (LOV) domain-containing proteins function as small light-activated modules capable of imparting blue light control of biological processes. Their small modular nature has made them model proteins for allosteric signal transduction and optogenetic devices. Despite intense research, key aspects of their signal transduction mechanisms and photochemistry remain poorly understood. In particular, ordered water has been identified as a possible key mediator of photocycle kinetics, ...[more]

PMID: 27226624

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Project description:BACKGROUND:The tropical ascomycete Trichoderma reesei (Hypocrea jecorina) represents one of the most efficient plant cell wall degraders. Regulation of the enzymes required for this process is affected by nutritional signals as well as other environmental signals including light. RESULTS:Our transcriptome analysis of strains lacking the photoreceptors BLR1 and BLR2 as well as ENV1 revealed a considerable increase in the number of genes showing significantly different transcript levels in light and darkness compared to wild-type. We show that members of all glycoside hydrolase families can be subject to light dependent regulation, hence confirming nutrient utilization including plant cell wall degradation as a major output pathway of light signalling. In contrast to N. crassa, photoreceptor mediated regulation of carbon metabolism in T. reesei occurs primarily by BLR1 and BLR2 via their positive effect on induction of env1 transcription, rather than by a presumed negative effect of ENV1 on the function of the BLR complex. Nevertheless, genes consistently regulated by photoreceptors in N. crassa and T. reesei are significantly enriched in carbon metabolic functions. Hence, different regulatory mechanisms are operative in these two fungi, while the light dependent regulation of plant cell wall degradation appears to be conserved.Analysis of growth on different carbon sources revealed that the oxidoreductive D-galactose and pentose catabolism is influenced by light and ENV1. Transcriptional regulation of the target enzymes in these pathways is enhanced by light and influenced by ENV1, BLR1 and/or BLR2. Additionally we detected an ENV1-regulated genomic cluster of 9 genes including the D-mannitol dehydrogenase gene lxr1, with two genes of this cluster showing consistent regulation in N. crassa. CONCLUSIONS:We show that one major output pathway of light signalling in Trichoderma reesei is regulation of glycoside hydrolase genes and the degradation of hemicellulose building blocks. Targets of ENV1 and BLR1/BLR2 are for the most part distinct and indicate individual functions for ENV1 and the BLR complex besides their postulated regulatory interrelationship.

Project description:Background:In addition to its outstanding cellulase production ability, Trichoderma reesei produces a wide variety of valuable secondary metabolites, the production of which has not received much attention to date. Among them, sorbicillinoids, a large group of hexaketide secondary metabolites derived from polyketides, are drawing a growing interest from researchers because they exhibit a variety of important biological functions, including anticancer, antioxidant, antiviral, and antimicrobial properties. The development of fungi strains with constitutive, hyperproduction of sorbicillinoids is thus desired for future industry application but is not well-studied. Moreover, although T. reesei has been demonstrated to produce sorbicillinoids with the corresponding gene cluster and biosynthesis pathway proposed, the underlying molecular mechanism governing sorbicillinoid biosynthesis remains unknown. Results:Recombinant T. reesei ZC121 was constructed from strain RUT-C30 by the insertion of the gene 12121-knockout cassette at the telomere of T. reesei chromosome IV in consideration of the off-target mutagenesis encountered during the unsuccessful deletion of gene 121121. Strain ZC121, when grown on cellulose, showed a sharp reduction of cellulase production, but yet a remarkable enhancement of sorbicillinoids production as compared to strain RUT-C30. The hyperproduction of sorbicillinoids is a constitutive process, independent of culture conditions such as carbon source, light, pH, and temperature. To the best of our knowledge, strain ZC121 displays record sorbicillinoid production levels when grown on both glucose and cellulose. Sorbicillinol and bisvertinolone are the two major sorbicillinoid compounds produced. ZC121 displayed a different morphology and markedly reduced sporulation compared to RUT-C30 but had a similar growth rate and biomass. Transcriptome analysis showed that most genes involved in cellulase production were downregulated significantly in ZC121 grown on cellulose, whereas remarkably all genes in the sorbicillinoid gene cluster were upregulated on both cellulose and glucose. Conclusion:A constitutive sorbicillinoid-hyperproduction strain T. reesei ZC121 was obtained by off-target mutagenesis, displaying an overwhelming shift from cellulase production to sorbicillinoid production on cellulose, leading to a record for sorbicillinoid production. For the first time, T. reesei degraded cellulose to produce platform chemical compounds other than protein in high yield. We propose that the off-target mutagenesis occurring at the telomere region might cause chromosome remodeling and subsequently alter the cell structure and the global gene expression pattern of strain ZC121, as shown by phenotype profiling and comparative transcriptome analysis of ZC121. Overall, T. reesei ZC121 holds great promise for the industrial production of sorbicillinoids and serves as a good model to explore the regulation mechanism of sorbicillinoids' biosynthesis.

Dataset Information

A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY.

Publications

A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY.

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