Biology subjects

Berggren, G.

Publications and source records attributed to Berggren, G..

5 recordsLinked to original sources

bioRxiv2024

Structure-guided engineering of α-ketoisocaproate dioxygenase increases isobutene production in Synechocystis sp. PCC 6803

Isobutene is a promising precursor for jet fuel due to its high energy density and favorable combustion properties. Light-driven bioproduction of isobutene has recently been investigated as an alternative strategy to crude oil refinement or fermentation-based manufacturing processes by harnessing the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the -ketoisocaproate dioxygenase (RnKICD) from Rattus norvegicus. However, the obtained production level was not sufficient, partially due the promiscuous activity of RnKICD. The enzyme catalyzes both the reaction with {rho}-hydroxyphenylpyruvate (HPP) for homogentisate formation, as well as the reaction with -ketoisocaproate (KIC), the precursor for isobutene synthesis. Here, to overcome this bottleneck step in the isobutene biosynthesis, protein engineering was employed to improve RnKICD activity and in vivo isobutene production. Purified RnKICD variants were characterized by measuring in vitro KIC and HPP consumption rates, as well as isobutene formation rate. The active site mutations F336V, N363A altered the KIC and HPP consumption rates, while the KIC-to-isobutene conversion ratio was only marginally affected. Besides, the RnKICD variants F336V, N363A and F336V/N363A exhibited a substantially enhanced substrate selectivity for KIC over HPP. Among the examined engineered Synechocystis strains, Syn-F336V showed a 4-fold improvement in isobutene production, compared to the base strain (Syn-RnKICD). Our findings reveal that residues F336 and N363 play a crucial role in substrate interactions, as targeted mutations at these sites shifted the substrate selectivity towards KIC and elevated the in vivo isobutene production levels significantly. We conclude that engineering the active site of RnKICD is a potent tool for improving isobutene bioproduction in Synechocystis. HighlightsO_LIPromiscuous -ketoisocaproate dioxygenase (KICD) from Rattus norvegicus was overproduced in Synechocystis sp. PCC 6803. C_LIO_LIProtein engineering was used to improve substrate selectivity. C_LIO_LIThe generated F336V variant led to a 4-fold increase in isobutene production in vivo. C_LI

bioengineering↗

bioRxiv2024

A widespread hydrogenase drives fermentative growth of gut bacteria in healthy people

Molecular hydrogen (H2) is among the most central, but least understood, metabolites in the human gastrointestinal tract (gut). H2 gas is produced in large quantities during bacterial fermentation and consumed as an energy source by bacteria and archaea. Disruption of H2 cycling is linked to gastrointestinal disorders, infections, and cancers, with H2 used as an indicator of gut dysfunction through breath tests. Despite this, the microorganisms, pathways, and enzymes mediating H2 production remain unresolved. Here we show that a previously uncharacterised enzyme, the group B [FeFe]-hydrogenase, drives most fermentative H2 production in the human gut. Analysis of stool, biopsy, and isolate (meta)genomes and (meta)transcriptomes show this hydrogenase is encoded by most gut bacteria and is highly expressed. Through analysis of 19 taxonomically diverse gut isolates, the group B [FeFe]-hydrogenase produces large amounts of H2 gas and supports fermentative growth of both Bacteroidetes and Firmicutes. Bacteroides particularly dominate H2 production. Biochemical and spectroscopic characterisation shows purified group B [FeFe]-hydrogenases are catalytically active and bind a di-iron active site. These hydrogenases are highly enriched in the guts of healthy individuals, but significantly depleted in favour of other fermentative hydrogenases in Crohns disease. Furthermore, we show that metabolically flexible respiratory bacteria are the most abundant H2 oxidizers in the gut, not sulfate reducers, methanogens, and acetogens as previously thought. This combination of enzymatic, cellular, and ecosystem-level analysis provides the first detailed understanding of H2 cycling in the human gut and reveals new links between microbiota function and gastrointestinal health.

microbiology↗

bioRxiv2022

Energy extraction from air: structural basis of atmospheric hydrogen oxidation

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival. This recently discovered yet globally significant process regulates the composition of the atmosphere, enhances soil biodiversity, and drives primary production in certain extreme environments. Atmospheric H2 oxidation has been attributed to still uncharacterised members of the [NiFe]-hydrogenase superfamily. However, it is unresolved how these enzymes overcome the extraordinary catalytic challenge of selectively oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2, and how the derived electrons are transferred to the respiratory chain. Here we determined the 1.52 [A] resolution CryoEM structure of the mycobacterial hydrogenase Huc and investigated its mechanism by integrating kinetics, electrochemistry, spectroscopy, mass spectrometry, and molecular dynamics simulations. Purified Huc is an oxygen-insensitive enzyme that couples the oxidation of atmospheric H2 at its large subunit to the hydrogenation of the respiratory electron carrier menaquinone at its small subunit. The enzyme uses a narrow hydrophobic gas channel to selectively bind atmospheric H2 at the expense of O2, while three [3Fe-4S] clusters and their unusual ligation by a D-histidine modulate the electrochemical properties of the enzyme such that atmospheric H2 oxidation is energetically feasible. Huc forms an 833 kDa complex composed of an octamer of catalytic subunits around a membrane-associated central stalk, which extracts and transports menaquinone a remarkable 94 [A] from the membrane, enabling its reduction. These findings provide a mechanistic basis for the biogeochemically and ecologically critical process of atmospheric H2 oxidation. Through the first characterisation of a group 2 [NiFe]-hydrogenase, we also uncover a novel mode of energy coupling dependent on long-range quinone transport and pave way for the development of biocatalysts that oxidize H2 in ambient air.

biochemistry↗

bioRxiv2018

Fusion of a functional glutaredoxin to the radical-generating subunit of ribonucleotide reductase

Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyse reduction of the four ribonucleotides to their corresponding deoxyribonucleotides. Facklamia ignava NrdB is an unprecedented fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-cone. Grx, which in general is encoded elsewhere in the genome than is the RNR operon, is a known physiological reductant of RNRs. Here we show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and interestingly also via a monothiol mechanism, although less efficiently. A Grx that utilizes either or of these two reaction mechanisms has to our knowledge not been observed with a native substrate before. The ATP-cone, which is commonly found as an N-terminal domain of the catalytic subunit of RNRs, is an allosteric on/off switch that promotes dNDP reduction in presence of ATP and inhibits the enzyme activity in presence of dATP. Here we show that dATP bound to the ATP-cone of F. ignava NrdB promotes formation of tetramers that are unable to form enzymatically competent complexes with F. ignava NrdA. The ATP-cone binds two molecules of dATP, but only one molecule of the activating nucleotide ATP. F. ignava NrdB contains the recently identified radical factor Mn2III/IV. We show that NrdA from the firmicute F. ignava can form a catalytically competent RNR with the Mn2III/IV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis.

biochemistry↗

bioRxiv2017

Unique ATP-cone-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit

Ribonucleotide reductases (RNRs) are key enzymes in DNA synthesis and repair, with sophisticated allosteric mechanisms controlling both substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leewenhoekiella blandensis radical-generating subunit regulates activity via modifications of quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzyme complexes. The tetramer forms solely by interactions between ATP-cones, as evidenced by a 2.45 [A] crystal structure. We also present evidence for an MnIIIMnIV metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by evolutionary capture of the domain by the radical-generating subunit. Our findings present a novel opportunity for dATP-regulation of engineered proteins.

biochemistry↗