Publications by authors named "Gregor Hoffmann"

2 Publications

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c-di-AMP assists osmoadaptation by regulating the potassium transporters KimA and KtrCD.

J Biol Chem 2019 11 9;294(44):16020-16033. Epub 2019 Sep 9.

Department of General Microbiology, Institute for Microbiology and Genetics, Georg-August University Göttingen, D-37077 Göttingen, Germany

Many bacteria and some archaea produce the second messenger cyclic diadenosine monophosphate (c-di-AMP). c-di-AMP controls the uptake of osmolytes in Firmicutes, including the human pathogen , making it essential for growth. c-di-AMP is known to directly regulate several potassium channels involved in osmolyte transport in species such as and , but whether this same mechanism is involved in , or even whether similar ion channels were present, was not known. Here, we have identified and characterized the putative potassium transporters KimA, KtrCD, and KdpABC. We demonstrate that expressing KimA and KtrCD, but not KdpABC, transport potassium into the cell, and both KimA and KtrCD are inhibited by c-di-AMP For KimA, c-di-AMP-dependent regulation requires the C-terminal domain. assays demonstrated that the dinucleotide binds to the cytoplasmic regulatory subunit KtrC and to the KdpD sensor kinase of the KdpDE two-component system, which in regulates the corresponding KdpABC transporter. Finally, we also show that contains a homolog of KimA, which mediates potassium transport. Thus, the c-di-AMP-dependent control of systems involved in potassium homeostasis seems to be conserved in phylogenetically related bacteria. Surprisingly, the growth of an mutant lacking the c-di-AMP-synthesizing enzyme is only weakly inhibited by potassium. Thus, the physiological impact of the c-di-AMP-dependent control of potassium uptake seems to be less pronounced in than in other Firmicutes.
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http://dx.doi.org/10.1074/jbc.RA119.010046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827311PMC
November 2019

Changing the substrate specificity of P450cam towards diphenylmethane by semi-rational enzyme engineering.

Protein Eng Des Sel 2011 May 27;24(5):439-46. Epub 2011 Jan 27.

Institute of Biochemistry, University of Leipzig, Deutscher Platz 5b, 04103 Leipzig, Germany.

A focused library comprising nine residues of the active site of P450cam monooxygenase resulting in ∼ 300,000 protein variants was screened for activity on diphenylmethane (DPM). The assay was based on the depletion of NADH by an in vitro reconstituted P450cam system in a 96-well scale. The throughput was increased by the parallel cultivation, purification and analysis of 20 variants per well (cluster screening). Thus ∼ 20,000 protein variants could be screened in summary of which five were found to transform DPM with a specific activity of up to 75% of the wild-type activity on d-camphor and a coupling rate of 7-18%. One variant converting DPM to 4-hydroxydiphenylmethane (4HDPM) was subjected to site-directed mutagenesis and saturation mutagenesis, which revealed the particular importance of positions F87, Y96 and L244 for substrate selectivity and the possibility for further improvements of this variant. Moreover, a reduction in size of the amino acid at position 396 decreased specific activity dramatically but increased coupling and switched the main product formation from 4HDPM towards diphenylmethanol.
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http://dx.doi.org/10.1093/protein/gzq119DOI Listing
May 2011