The structural basis for seryl-adenylate and Ap4A synthesis by seryl-tRNA synthetase

AbstractBackground: Seryl-tRNA synthetase is a homodimeric class II aminoacyl-tRNA synthetase that specifically charges cognate tRNAs with serine. In the first step of this two-step reaction, Mg·ATP and serine react to form the activated intermediate, seryl-adenylate. The serine is subsequently transferred to the 3′-end of the tRNA. In common with most other aminoacyl-tRNA synthetases, seryl-tRNA synthetase is capable of synthesizing diadenosine tetraphosphate (Ap4A) from the enzyme-bound adenylate intermediate and a second molecule of ATP. Understanding the structural basis for the substrate specificity and the catalytic mechanism of aminoacyl-tRNA synthetases is of considerable general interest because of the fundamental importance of these enzymes to protein biosynthesis in all living cells.Results Crystal structures of three complexes of seryl-tRNA synthetase from Thermus thermophilus are described. The first complex is of the enzyme with ATP and Mn2+. The ATP is found in an unusual bent conformation, stabilized by interactions with conserved arginines and three manganese ions. The second complex contains seryl-adenylate in the active site, enzymatically produced in the crystal after soaking with ATP, serine and Mn2+. The third complex is between the enzyme, Ap4A and Mn2+. All three structures exhibit a common Mn2+ site in which the cation is coordinated by two active-site residues in addition to the α-phosphate group from the bound ligands.Conclusion Superposition of these structures allows a common reaction mechanism for seryl-adenylate and Ap4A formation to be proposed. The bent conformation of the ATP and the position of the serine are consistent with nucleophilic attack of the serine carboxyl group on the α-phosphate by an in-line displacement mechanism leading to the release of the inorganic pyrophosphate. A second ATP molecule can bind with its γ-phosphate group in the same position as the β-phosphate of the original ATP. This can attack the seryl-adenylate with the formation of Ap4A by an identical in-line mechanism in the reverse direction. The divalent cation is essential for both reactions and may be directly involved in stabilizing the transition state

T Cell Recognition of the Dominant I-Ak–Restricted Hen Egg Lysozyme Epitope: Critical Role for Asparagine Deamidation

Type-B T cells raised against the immunodominant peptide in hen egg lysozyme (HEL48–62) do not respond to whole lysozyme, and this has been thought to indicate that peptide can bind to l-Ak in different conformations. Here we demonstrate that such T cells recognize a deamidated form of the HEL peptide and not the native peptide. The sequence of the HEL epitope facilitates rapid and spontaneous deamidation when present as a free peptide or within a flexible domain. However, this deamidated epitope is not created within intact lysozyme, most likely because it resides in a highly structured part of the protein. These findings argue against the existence of multiple conformations of the same peptide–MHC complex and have important implications for the design of peptide-based vaccines. Furthermore, as the type-B T cells are known to selectively evade induction of tolerance when HEL is expressed as a transgene, these results suggest that recognition of posttranslationally modified self-antigen may play a role in autoimmunity

How coenzyme B12 radicals are generated: the crystal structure of methylmalonyl-coenzyme A mutase at 2 å resolution

AbstractBackground: The enzyme methylmalonyl-coenzyme A (CoA) mutase, an αβ heterodimer of 150 kDa, is a member of a class of enzymes that uses coenzyme B12 (adenosylcobalamin) as a cofactor. The enzyme induces the formation of an adenosyl radical from the cofactor. This radical then initiates a free-radical rearrangement of its substrate, succinyl-CoA, to methylmalonyl-CoA.Results Reported here is the crystal structure at 2 å resolution of methylmalonyl-CoA mutase from Propionibacterium shermanii in complex with coenzyme B12 and with the partial substrate desulpho-CoA (lacking the succinyl group and the sulphur atom of the substrate). The coenzyme is bound by a domain which shares a similar fold to those of flavodoxin and the B12-binding domain of methylcobalamin-dependent methionine synthase. The cobalt atom is coordinated, via a long bond, to a histidine from the protein. The partial substrate is bound along the axis of a (β/α)8 TIM barrel domain.Conclusion The histidine–cobalt distance is very long (2.5 å compared with 1.95–2.2 å in free cobalamins), suggesting that the enzyme positions the histidine in order to weaken the metal–carbon bond of the cofactor and favour the formation of the initial radical species. The active site is deeply buried, and the only access to it is through a narrow tunnel along the axis of the TIM barrel domain

Anomaly Analysis in Cleaning-in-Place Operations of an Industrial Brewery Fermenter

Analyzing historical data of industrial cleaning-in-place (CIP) operations is essential to avoid potential operation failures but is usually not done. This paper presents a three-level approach of analysis based on the CIP case of a brewery fermenter to describe how to analyze the historical data in steps for detecting anomalies. In the first level, the system is assessed before cleaning to ensure that the selected recipe and system are able to accomplish the task. In the second level, a multiway principal component analysis (MPCA) algorithm is applied to monitor the process variables online or post cleaning, with the purpose of locally detecting the anomalies and explaining the potential causes of the anomalous event. The third level analysis is performed after cleaning to evaluate the cleaning results. The implementation of the analysis approach has significant potential to automatically detect deviations and anomalies in future CIP cycles and to optimize the cleaning process