Structure of the E. coli agmatinase, SPEB.

Agmatine amidinohydrolase, or agmatinase, catalyzes the conversion of agmatine to putrescine and urea. This enzyme is found broadly across kingdoms of life and plays a critical role in polyamine biosynthesis and the regulation of agmatine concentrations. Here we describe the high-resolution X-ray crystal structure of the E. coli agmatinase, SPEB. The data showed a relatively high degree of pseudomerohedral twinning, was ultimately indexed in the P31 space group and led to a final model with eighteen chains, corresponding to three full hexamers in the asymmetric unit. There was a solvent content of 38.5% and refined R/Rfree values of 0.166/0.216. The protein has the conserved fold characteristic of the agmatine ureohydrolase family and displayed a high degree of structural similarity among individual protomers. Two distinct peaks of electron density were observed in the active site of most of the eighteen chains of SPEB. As the activity of this protein is known to be dependent upon manganese and the fold is similar to other dinuclear metallohydrolases, these peaks were modeled as manganese ions. The orientation of the conserved active site residues, in particular those amino acids that participate in binding the metal ions and a pair of acidic residues (D153 and E274 in SPEB) that play a role in catalysis, are similar to other agmatinase and arginase enzymes and is consistent with a hydrolytic mechanism that proceeds via a metal-activated hydroxide ion.

Structural Basis for the Regulation of Biofilm Formation and Iron Uptake in A. baumannii by the Blue-Light-Using Photoreceptor, BlsA.

The opportunistic human pathogen, A. baumannii, senses and responds to light using the blue light sensing A (BlsA) photoreceptor protein. BlsA is a blue-light-using flavin adenine dinucleotide (BLUF) protein that is known to regulate a wide variety of cellular functions through interactions with different binding partners. Using immunoprecipitation of tagged BlsA in A. baumannii lysates, we observed a number of proteins that interact with BlsA, including several transcription factors. In addition to a known binding partner, the iron uptake regulator Fur, we identified the biofilm response regulator BfmR as a putative BlsA-binding partner. Using microscale thermophoresis, we determined that both BfmR and Fur bind to BlsA with nanomolar binding constants. To better understand how BlsA interacts with and regulates these transcription factors, we solved the X-ray crystal structures of BlsA in both a ground (dark) state and a photoactivated light state. Comparison of the light- and dark-state structures revealed that, upon photoactivation, the two α-helices comprising the variable domain of BlsA undergo a distinct conformational change. The flavin-binding site, however, remains largely unchanged from dark to light. These structures, along with docking studies of BlsA and Fur, reveal key mechanistic details about how BlsA propagates the photoactivation signal between protein domains and on to its binding partner. Taken together, our structural and biophysical data provide important insights into how BlsA controls signal transduction in A. baumannii and provides a likely mechanism for blue-light-dependent modulation of biofilm formation and iron uptake.

Crystal structure of E. coli PRPP synthetase.

BACKGROUND: Ribose-phosphate pyrophosphokinase (EC 2.7.6.1) is an enzyme that catalyzes the ATP-dependent conversion of ribose-5-phosphate to phosphoribosyl pyrophosphate. The reaction product is a key precursor for the biosynthesis of purine and pyrimidine nucleotides. RESULTS: We report the 2.2 Å crystal structure of the E. coli ribose-phosphate pyrophosphobinase (EcKPRS). The protein has two type I phosphoribosyltransferase folds, related by 2-fold pseudosymmetry. The propeller-shaped homohexameric structure of KPRS is composed of a trimer of dimers, with the C-terminal domains forming the dimeric blades of the propeller and the N-terminal domains forming the hexameric core. The key, conserved active site residues are well-defined in the structure and positioned appropriately to bind substrates, adenosine monophosphate and ribose-5-phosphate. The allosteric site is also relatively well conserved but, in the EcKPRS structure, several residues from a flexible loop occupy the site where the allosteric modulator, adenosine diphosphate, is predicted to bind. The presence of the loop in the allosteric site may be an additional level of regulation, whereby low affinity molecules are precluded from binding. CONCLUSIONS: Overall, this study details key structural features of an enzyme that catalyzes a critical step in nucleotide metabolism. This work provides a framework for future studies of this important protein and, as nucleotides are critical for viability, may serve as a foundation for the development of novel anti-bacterial drugs.

Higher order structures in purine and pyrimidine metabolism.

The recent discovery of several forms of higher order protein structures in cells has shifted the paradigm of how we think about protein organization and metabolic regulation. These dynamic and controllable protein assemblies, which are composed of dozens or hundreds of copies of an enzyme or related enzymes, have emerged as important players in myriad cellular processes. We are only beginning to appreciate the breadth of function of these types of macromolecular assemblies. These higher order structures, which can be assembled in response to varied cellular stimuli including changing metabolite concentrations or signaling cascades, give the cell the capacity to modulate levels of biomolecules both temporally and spatially. This provides an added level of control with distinct kinetics and unique features that can be harnessed as a subtle, yet powerful regulatory mechanism. Due, in large part, to advances in structural methods, such as crystallography and cryo-electron microscopy, and the advent of super-resolution microscopy techniques, a rapidly increasing number of these higher order structures are being identified and characterized. In this review, we detail what is known about the structure, function and control mechanisms of these mesoscale protein assemblies, with a particular focus on those involved in purine and pyrimidine metabolism. These structures have important implications both for our understanding of fundamental cellular processes and as fertile ground for new targets for drug discovery and development.