Measuring carbonic anhydrase activity in alpha-carboxysomes using stopped-flow.

Carboxysomes are protein-based organelles that serve as the centerpiece of the bacterial CO concentration mechanism (CCM). They are present in all cyanobacteria and many chemoautotrophic proteobacteria and encapsulate the key enzymes for CO fixation, carbonic anhydrase and the carboxylase Rubisco, within a protein shell. The CCM actively accumulates bicarbonate in the cytosol, which diffuses into the carboxysome where carbonic anhydrase rapidly equilibrates it to CO.

Identification of a carbonic anhydrase-Rubisco complex within the alpha-carboxysome.

Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO fixation-Rubisco and carbonic anhydrase-and are the centerpiece of the bacterial CO concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO by carbonic anhydrase, producing a high CO concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the α-carboxysome.

Rubisco forms a lattice inside alpha-carboxysomes.

Despite the importance of microcompartments in prokaryotic biology and bioengineering, structural heterogeneity has prevented a complete understanding of their architecture, ultrastructure, and spatial organization. Here, we employ cryo-electron tomography to image α-carboxysomes, a pseudo-icosahedral microcompartment responsible for carbon fixation. We have solved a high-resolution subtomogram average of the Rubisco cargo inside the carboxysome, and determined the arrangement of the enzyme.

Functional reconstitution of a bacterial CO concentrating mechanism in .

Many photosynthetic organisms employ a CO concentrating mechanism (CCM) to increase the rate of CO fixation via the Calvin cycle. CCMs catalyze ≈50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an strain engineered to depend on rubisco carboxylation for growth.

Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate.

DABs are inorganic carbon pumps found throughout prokaryotic phyla.

Bacterial autotrophs often rely on CO concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, a systematic screen of the components of CCMs is lacking. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the γ-proteobacterium Halothiobacillus neapolitanus.

Systematic identification of recognition motifs for the hub protein LC8.

Hub proteins participate in cellular regulation by dynamic binding of multiple proteins within interaction networks. The hub protein LC8 reversibly interacts with more than 100 partners through a flexible pocket at its dimer interface. To explore the diversity of the LC8 partner pool, we screened for LC8 binding partners using a proteomic phage display library composed of peptides from the human proteome, which had no bias toward a known LC8 motif.

High-throughput methods for identification of protein-protein interactions involving short linear motifs.

Interactions between modular domains and short linear motifs (3-10 amino acids peptide stretches) are crucial for cell signaling. The motifs typically reside in the disordered regions of the proteome and the interactions are often transient, allowing for rapid changes in response to changing stimuli. The properties that make domain-motif interactions suitable for cell signaling also make them difficult to capture experimentally and they are therefore largely underrepresented in the known protein-protein interaction networks.

Substrate scope and selectivity in offspring to an enzyme subjected to directed evolution.

We have analyzed the effects of mutations inserted during directed evolution of a specialized enzyme, Escherichia coli S-1,2-propanediol oxidoreductase (FucO). The kinetic properties of evolved variants have been determined and the observed differences have been rationalized by modeling the tertiary structures of isolated variants and the wild-type enzyme. The native substrate, S-1,2-propanediol, as well as phenylacetaldehyde and 2S-3-phenylpropane-1,2-diol, which are new substrates accepted by isolated variants, were docked into the active sites.

Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway.

Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate.

FtsZ degradation in the cyanobacterium Anabaena sp. strain PCC 7120.

In prokaryotes, cell division is normally achieved by binary fission, and the key player FtsZ is considered essential for the complete process. In cyanobacteria, much remains unknown about several aspects of cell division, including the identity and mechanism of the various components involved in the division process. Here, we report results obtained from a search of the players implicated in cell division, directly associating to FtsZ in the filamentous, heterocyst-forming cyanobacterium Anabaena sp.

Emergence of a novel highly specific and catalytically efficient enzyme from a naturally promiscuous glutathione transferase.

Redesign of glutathione transferases (GSTs) has led to enzymes with remarkably enhanced catalytic properties. Exchange of substrate-binding residues in GST A1-1 created a GST A4-4 mimic, called GIMFhelix, with >300-fold improved activity with nonenal and suppressed activity with other substrates. In the present investigation GIMFhelix was compared with the naturally-evolved GSTs A1-1 and A4-4 by determining catalytic efficiencies with nine alternative substrates.