Reliance on glutamine has long been considered a hallmark of cancer cell metabolism. However, some recent studies have challenged this notion in vivo, prompting a need for further clarifications on the role of glutamine metabolism in cancer. We find that there is ample evidence of an essential role for glutamine in tumors and that a variety of factors, including tissue type, the underlying cancer genetics, the tumor microenvironment and other variables such as diet and host physiology collectively influence the role of glutamine in cancer. Thus the requirements for glutamine in cancer are overall highly heterogeneous. In this review, we discuss the implications both for basic science and for targeting glutamine metabolism in cancer therapy.

The enterobacterium Escherichia coli synthesizes two H 2 uptake enzymes, Hyd-1 and Hyd-2. We show using precise electrochemical kinetic measurements that the properties of Hyd-1 and Hyd-2 contrast strikingly, and may be individually optimized to function under distinct environmental conditions. Hyd-2 is well suited for fast and efficient catalysis in more reducing environments, to the extent that in vitro it behaves as a bidirectional hydrogenase. In contrast, Hyd-1 is active for H 2 oxidation under more oxidizing conditions and cannot function in reverse. Importantly, Hyd-1 is O 2 tolerant and can oxidize H 2 in the presence of air, whereas Hyd-2 is ineffective for H 2 oxidation under aerobic conditions. The results have direct relevance for physiological roles of Hyd-1 and Hyd-2, which are expressed in different phases of growth. The properties that we report suggest distinct technological applications of these contrasting enzymes.Hydrogenases catalyze the reversible cleavage of H 2 into protons and electrons, and play an important role in the energy metabolism of a broad range of microorganisms (1). Hydrogenases are classified according to their active site metal ion content, and three phylogenetically distinct classes have so far been identified: di-iron [FeFe]-, nickel-iron [NiFe]-, and mono-iron [Fe]-hydrogenases (1). Nickel-iron hydrogenases are the most abundant of the three types (1), and many members of this class are membrane bound, with the membrane-extrinsic domain consisting of a large subunit containing the active site, and a small subunit accommodating one to three electron-transferring iron-sulfur clusters. The active sites of [NiFe]-hydrogenases contain a nickel atom coordinated by four cysteine-S ligands, two of which bridge to an iron atom that is further coordinated by three unusual diatomic ligands, two cyanides and one carbonyl (2).Hydrogenases are inactivated by O 2

The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism.

An important clue to the mechanism for O(2) tolerance of certain [NiFe]-hydrogenases is the conserved presence of a modified environment around the iron-sulfur cluster that is proximal to the active site. The O(2)-tolerant enzymes contain two cysteines, located at opposite ends of this cluster, which are glycines in their O(2)-sensitive counterparts. The strong correlation highlights special importance for electron-transfer activity in the protection mechanism used to combat O(2). Site-directed mutagenesis has been carried out on Escherichia coli hydrogenase-1 to substitute these cysteines (C19 and C120) individually and collectively for glycines, and the effects of each replacement have been determined using protein film electrochemistry and electron paramagnetic resonance (EPR) spectroscopy. The "split" iron-sulfur cluster EPR signal thus far observed when oxygen-tolerant [NiFe]-hydrogenases are subjected to oxidizing potentials is found not to provide any simple, reliable correlation with oxygen tolerance. Oxygen tolerance is largely conferred by a single cysteine (C19), replacement of which by glycine removes the ability to function even in 1% O(2).

To support sustained biomass accumulation, tumor cells undergo metabolic reprogramming. Nutrient transporters and metabolic enzymes are regulated by the same oncogenic signals that drive cell-cycle progression. Some of the earliest cancer therapies used antimetabolites to disrupt tumor metabolism, and there is now renewed interest in developing drugs that target metabolic dependencies. Many cancers exhibit increased demand for specific amino acids, and become dependent on either an exogenous supply or upregulated de novo synthesis. Strategies to exploit such ‘metabolic addictions’ include depleting amino acids in blood serum, blocking uptake by transporters and inhibiting biosynthetic or catabolic enzymes. Recent findings highlight the importance of using appropriate model systems and identifying target patient groups as potential therapies advance into the clinic.

Many cancer cells exhibit an altered metabolic phenotype, in which glutamine consumption is upregulated relative to healthy cells. This metabolic reprogramming often depends upon mitochondrial glutaminase activity, which converts glutamine to glutamate, a key precursor for biosynthetic and bioenergetic processes. Two isozymes of glutaminase exist, a kidney-type (GLS) and a liver-type enzyme (GLS2 or LGA). While a majority of studies have focused on GLS, here we summarize key findings on both glutaminases, describing their structure and function, their roles in cancer and pharmacological approaches to inhibiting their activities.

"Hyd-1", produced by Escherichia coli , exemplifies a special class of [NiFe]-hydrogenase that can sustain high catalytic H(2) oxidation activity in the presence of O(2)-an intruder that normally incapacitates the sulfur- and electron-rich active site. The mechanism of "O(2) tolerance" involves a critical role for the Fe-S clusters of the electron relay, which is to ensure the availability-for immediate transfer back to the active site-of all of the electrons required to reduce an attacking O(2) molecule completely to harmless H(2)O. The unique [4Fe-3S] cluster proximal to the active site is crucial because it can rapidly transfer two of the electrons needed. Here we investigate and establish the equally crucial role of the high potential medial [3Fe-4S] cluster, located >20 Å from the active site. A variant, P242C, in which the medial [3Fe-4S] cluster is replaced by a [4Fe-4S] cluster, is unable to sustain steady-state H(2) oxidation activity in 1% O(2). The [3Fe-4S] cluster is essential only for the first stage of complete O(2) reduction, ensuring the supply of all three electrons needed to form the oxidized inactive state "Ni-B" or "Ready" (Ni(III)-OH). Potentiometric titrations show that Ni-B is easily reduced (E(m) ≈ +0.1 V at pH 6.0); this final stage of the O(2)-tolerance mechanism regenerates active enzyme, effectively completing a competitive four-electron oxidase cycle and is fast regardless of alterations at the proximal or medial clusters. As a consequence of all these factors, the enzyme's response to O(2), viewed by its electrocatalytic activity in protein film electrochemistry (PFE) experiments, is merely to exhibit attenuated steady-state H(2) oxidation activity; thus, O(2) behaves like a reversible inhibitor rather than an agent that effectively causes irreversible inactivation. The data consolidate a rich picture of the versatile role of Fe-S clusters in electron relays and suggest that Hyd-1 can function as a proficient hydrogen oxidase.

PAGE 17257:Fig. 2, line 9 should read: F, a projection through a 350 nm thick section of longitudinally sectioned P3H1 null tendon from which the tilt series (supplemental Fig. S1) was collected. ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.orgWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.