A metallocofactor containing iron, sulfur, copper, and nickel has been discovered in the enzyme carbon monoxide dehydrogenase/acetyl-CoA (coenzyme A) synthase from Moorella thermoacetica (f. Clostridium thermoaceticum ). Our structure at 2.2 angstrom resolution reveals that the cofactor responsible for the assembly of acetyl-CoA contains a [Fe ₄ S ₄ ] cubane bridged to a copper-nickel binuclear site. The presence of these three metals together in one cluster was unanticipated and suggests a newly discovered role for copper in biology. The different active sites of this bifunctional enzyme complex are connected via a channel, 138 angstroms long, that provides a conduit for carbon monoxide generated at the C-cluster on one subunit to be incorporated into acetyl-CoA at the A-cluster on the other subunit.

Some like it hot, others not: Enzymes strike a delicate balance between features that enhance chemical reactivity and those that contribute to stable structure. Both features are important and can be unrelated or antagonistic. Pinneyet al.combined rich experimental work on thermophilic and mesophilic variants of the enzyme ketosteroid isomerase (KSI) with bioinformatic data from a diverse set of bacterial enzymes to reveal the molecular determinants of thermal adaptation in enzymes. For KSI, they observed a trade-off between activity and thermal stability that comes down to a single active-site residue. With their larger dataset, they identified patterns of individual amino acid substitutions that are favored at higher temperatures, and also consider how networks of stabilizing interactions develop.Science, this issue p.eaay2784

POT1 and TPP1 are part of the shelterin complex and are essential for telomere length regulation and maintenance. Naturally occurring mutations of the telomeric POT1–TPP1 complex are implicated in familial glioma, melanoma and chronic lymphocytic leukaemia. Here we report the atomic structure of the interacting portion of the human telomeric POT1–TPP1 complex and suggest how several of these mutations contribute to malignant cancer. The POT1 C-terminus (POT1C) forms a bilobal structure consisting of an OB-fold and a holiday junction resolvase domain. TPP1 consists of several loops and helices involved in extensive interactions with POT1C. Biochemical data shows that several of the cancer-associated mutations, partially disrupt the POT1–TPP1 complex, which affects its ability to bind telomeric DNA efficiently. A defective POT1–TPP1 complex leads to longer and fragile telomeres, which in turn promotes genomic instability and cancer.

How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data for Pseudomonas putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes. Significance Enzymes position catalytic and reactant groups in active sites, but motions are also integral to enzyme function. Decades of research have revealed static snapshots for many enzymes, but we know very little about the extents and directions of motions of the residues that make up enzymes and their active sites. We took advantage of emerging crystallographic approaches to provide information about protein conformational ensembles for the enzyme ketosteroid isomerase (KSI). Ensemble analyses indicated limited change in conformational heterogeneity through the enzyme’s reaction cycle and a balance of positioning and flexibility that allows KSI to efficiently catalyze multiple reaction steps. We tested KSI-specific and general models for enzyme catalysis, demonstrating the power of and need for “ensemble–function” interrogation of enzymes.