Biology subjects

Krishna, R. N.

Publications and source records attributed to Krishna, R. N..

3 recordsLinked to original sources

bioRxiv2019

Structural control of regioselectivity in an unusual bacterial acyl-CoA dehydrogenase

Terminal alkenes are easily derivatized, making them desirable functional group targets for polyketide synthase (PKS) engineering. However, they are rarely encountered in natural PKS systems. One mechanism for terminal alkene formation in PKSs is through the activity of an acyl-CoA dehydrogenase (ACAD). Herein, we use biochemical and structural analysis to understand the mechanism of terminal alkene formation catalyzed by an {gamma},{delta}-ACAD from the biosynthesis of the polyketide natural product FK506, TcsD. While TcsD is homologous to canonical ,{beta}-ACADs, it acts regioselectively at the {gamma},{delta}-position and only on ,{beta}-unsaturated substrates. Furthermore, this regioselectivity is controlled by a combination of bulky residues in the active site and a lateral shift in the positioning of the FAD cofactor within the enzyme. Substrate modeling suggests that TcsD utilizes a novel set of hydrogen bond donors for substrate activation and positioning, preventing dehydrogenation at the ,{beta} position of substrates. From the structural and biochemical characterization of TcsD, key residues that contribute to regioselectivity and are unique to the protein family were determined and used to identify other putative {gamma},{delta}-ACADs that belong to diverse natural product biosynthetic gene clusters. These predictions are supported by the demonstration that a phylogenetically distant homolog of TcsD also regioselectively oxidizes ,{beta}-unsaturated substrates. This work exemplifies a powerful approach to understand unique enzymatic reactions and will facilitate future enzyme discovery, inform enzyme engineering, and aid natural product characterization efforts.

biochemistry↗

bioRxiv2019

Glutarate metabolism in Pseudomonas putida is regulated by two distinct glutarate sensing transcription factors

Transcription factor based biosensors can be leveraged to screen thousands of genetics designs for optimal production in engineered microbes. In this study we characterize two glutarate sensing transcription factors (CsiR and GcdR) from Pseudomonas putida. The genomic contexts of CsiR homologs were analyzed and DNA binding sites were bioinformatically predicted. Both CsiR and GcdR were purified and shown to bind upstream of their coding sequencing in vitro. CsiR was shown to dissociate from DNA in vitro when exogenous glutarate was added confirming it acts as a genetic repressor. Both transcription factors were then engineered into plasmid based biosensors and their respective sensing performance features calculated. Both sensor plasmids were then reintroduced into P. putida and were evaluated for their ability to sense flux through glutarate when grown on various lysine metabolites as sole carbon sources. These findings provide information describing glutarate flux in P. putida, and potentially useful tools for future metabolic engineering and synthetic biology efforts.

microbiology↗

bioRxiv2018

Massively parallel fitness profiling reveals multiple novel enzymes in Pseudomonas putida lysine metabolism

The lysine metabolism of Pseudomonas putida can produce multiple important commodity chemicals and is implicated in rhizosphere colonization. However, despite intensive study, the biochemical and genetic links between lysine metabolism and central metabolism remain unresolved in P. putida. Here, we leverage Random Barcode Transposon Sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both L- and D-lysine metabolism. We first describe three pathway enzymes that catabolize 2-aminoadipate (2AA) to 2-ketoglutarate (2KG) connecting D-lysine to the TCA cycle. One of these enzymes, PP_5260, contains a DUF1338 domain, a family without a previously described biological function. We demonstrate PP_5260 converts 2-oxoadipate (2OA) to 2-hydroxyglutarate (2HG), a novel biochemical reaction. We expand on recent work showing that the glutarate hydroxylase, CsiD, can co-utilize both 2OA and 2KG as a co-substrate in the hydroxylation of glutarate. Finally we demonstrate that the cellular abundance of D- and L-lysine pathway proteins are highly sensitive to pathway specific substrates. This work demonstrates the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria.\n\nImportanceO_LIP. putida is an attractive host for metabolic engineering as its lysine metabolism can be utilized for the production of multiple important commodity chemicals.\nC_LIO_LIWe demonstrate the first biochemical evidence of a bacterial 2OA catabolic pathway to central metabolites.\nC_LIO_LIDUF1338 proteins are widely dispersed across many kingdoms of life. Here we demonstrate the first biochemical evidence of function for a member of this protein family.\nC_LI

microbiology↗