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Mevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high‐resolution X‐ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N‐terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases. [ABSTRACT FROM AUTHOR]

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins. [ABSTRACT FROM AUTHOR]

Ssr4 is a yeast protein from Schizosaccharomyces pombe and is an essential part of the chromatin‐remodelling [SWI/SNF and RSC (remodelling the structure of chromatin)] complexes found in S. pombe. These complexes (or their homologues) regulate gene expression in eukaryotic organisms, affecting a large number of genes both positively and negatively. The downstream effects are seen in development, and in humans have implications for disease such as cancer. The chromatin structure is altered by modifying the DNA–histone contacts, thus opening up or closing down sections of DNA to specific transcription factors that regulate the transcription of genes. The Ssr4 sequence has little homology to other sequences in the Protein Data Bank, so the structure was solved using an iodine derivative with SAD phasing. The structure of the N‐terminal domain is an antiparallel β‐sheet of seven strands with α‐helices on one side and random coil on the other. The structure is significantly different to deposited structures and was used as a target in the most recent Critical Assessment of Techniques for Protein Structure Prediction (CASP; https://predictioncenter.org/) competition. [ABSTRACT FROM AUTHOR]

Cancer is one of the leading causes of mortality in humans, and recent work has focused on the area of immuno‐oncology, in which the immune system is used to specifically target cancerous cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an emerging therapeutic target in human cancers owing to its role in degrading cyclic GMP‐AMP (cGAMP), an agonist of the stimulator of interferon genes (STING). The available structures of ENPP1 are of the mouse enzyme, and no structures are available with anything other than native nucleotides. Here, the first X‐ray crystal structures of the human ENPP1 enzyme in an apo form, with bound nucleotides and with two known inhibitors are presented. The availability of these structures and a robust crystallization system will allow the development of structure‐based drug‐design campaigns against this attractive cancer therapeutic target. [ABSTRACT FROM AUTHOR]

Biuret deamination is an essential step in cyanuric acid mineralization. In the well-studied atrazine degrading bacterium Pseudomonas sp. strain ADP, the amidase AtzE catalyzes this step. However, Rhizobium leguminosarum bv. viciae 3841 uses an unrelated cysteine hydrolase, BiuH, instead. Herein, structures of BiuH, BiuH with bound inhibitor and variants of BiuH are reported. The substrate is bound in the active site by a hydrogen bonding network that imparts high substrate specificity. The structure of the inactive Cys175Ser BiuH variant with substrate bound in the active site revealed that an active site cysteine (Cys175), aspartic acid (Asp36) and lysine (Lys142) form a catalytic triad, which is consistent with biochemical studies of BiuH variants. Finally, molecular dynamics simulations highlighted the presence of three channels from the active site to the enzyme surface: a persistent tunnel gated by residues Val218 and Gln215 forming a potential substrate channel and two smaller channels formed by Val28 and a mobile loop (including residues Phe41, Tyr47 and Met51) that may serve as channels for co-product (ammonia) or co-substrate (water). [ABSTRACT FROM AUTHOR]

The N-isopropylammelide isopropylaminohydrolase from Pseudomonas sp. strain ADP, AtzC, provides the third hydrolytic step in the mineralization of s-triazine herbicides, such as atrazine. We obtained the X-ray crystal structure of AtzC at 1.84 Å with a weak inhibitor bound in the active site and then used a combination of in silico docking and site-directed mutagenesis to understand the interactions between AtzC and its substrate, isopropylammelide. The substitution of an active site histidine residue (His249) for an alanine abolished the enzyme’s catalytic activity. We propose a plausible catalytic mechanism, consistent with the biochemical and crystallographic data obtained that is similar to that found in carbonic anhydrase and other members of subtype III of the amidohydrolase family [ABSTRACT FROM AUTHOR]

Although part of the coenzyme A pathway, vanin 1 (also known as pantetheinase) sits on the cell surface of many cell types as an ectoenzyme, catalyzing the breakdown of pantetheine to pantothenic acid (vitamin B5) and cysteamine, a strong reducing agent. Vanin 1 was initially discovered as a protein involved in the homing of leukocytes to the thymus. Numerous studies have shown that vanin 1 is involved in inflammation, and more recent studies have shown a key role in metabolic disease. Here, the X-ray crystal structure of human vanin 1 at 2.25 Å resolution is presented, which is the first reported structure from the vanin family, as well as a crystal structure of vanin 1 bound to a specific inhibitor. These structures illuminate how vanin 1 can mediate its biological roles by way of both enzymatic activity and protein-protein interactions. Furthermore, it sheds light on how the enzymatic activity is regulated by a novel allosteric mechanism at a domain interface. [ABSTRACT FROM AUTHOR]

The REMARK280 field of the Protein Data Bank is the richest open source of successful crystallization information. The REMARK280 field is optional and currently uncurated, so significant effort needs to be applied to extract reliable data. There are well over 15 000 crystallization conditions available commercially from 12 different vendors. After putting the PDB crystallization information and the commercial cocktail data into a consistent format, these data are used to extract information about the overlap between the two sets of crystallization conditions. An estimation is made as to which commercially available conditions are most appropriate for producing well diffracting crystals by looking at which commercial conditions are found unchanged (or almost unchanged) in the PDB. Further analyses include which commercial kits are the most appropriate for shotgun or more traditional approaches to crystallization screening. This analysis suggests that almost 40% of the crystallization conditions found currently in the PDB are identical or very similar to a commercial condition. [ABSTRACT FROM AUTHOR]

We present a new approach to carbonicanhydrase II (CA II) inhibitordesign that enables close interrogation of the regions of the CA activesite where there is the greatest variability in amino acid residuesamong the different CA isozymes. By appending dual tail groups ontothe par excellenceCA inhibitor acetazolamide, compoundsthat may interact with the distinct hydrophobic and hydrophilic halvesof the CA II active site were prepared. The dual-tail combinationsselected included (i) two hydrophobic moieties, (ii) two hydrophilicmoieties, and (iii) one hydrophobic and one hydrophilic moiety. TheCA enzyme inhibition profile as well as the protein X-ray crystalstructure of compound 3, comprising one hydrophobic andone hydrophilic tail moiety, in complex with CA II is described. Thisnovel dual-tail approach has provided an enhanced opportunity to morefully exploit interactions with the CA active site by enabling thesemolecules to interact with the distinct halves of the active site.In addition to the dual-tail compounds, a corresponding set of single-tailderivatives was synthesized, enabling a comparative analysis of thesingle-tail versus dual-tail compound CA inhibition profile. [ABSTRACT FROM AUTHOR]

Obtaining well-diffracting crystals of membrane proteins, which is crucial to the molecular-level understanding of their intrinsic three-dimensional structure, dynamics, and function, represents a fundamental bottleneck in the field of structural biology. One of the major advances in the field of membrane protein structural determination was the realization that the nanostructured lipidic cubic phase (LCP) environment constitutes a membrane mimetic matrix that promotes solubilization, stabilization, and crystallization of specific membrane proteins. Despite two decades passing since the introduction of LCP-based membrane protein crystallization, the research community's understanding of the processes that drive protein nucleation and macromolecular assembly in the LCP generally remains limited. In the current study, we present a detailed, systematic investigation into the relationship between the chemical structure of the lipid, the physical properties of the ensuing mesophase, the translational diffusion of the encapsulated membrane protein, and the resulting protein crystallization. Importantly, we show for the first time that cubic phase transport properties directly modulate the size and morphology of fully grown protein crystals without affecting their crystallographic space group. These findings provide a deeper understanding of the LCP-based crystal growth process, setting the stage for the engineering of membrane protein crystals at the molecular level via intentional design of the host cubic phase, and, ultimately, accelerating progress in the field of membrane protein structural biology. [ABSTRACT FROM AUTHOR]