No abstract

Structural snapshots of each step in the catalytic cycle would help development of inhibitors of human immunodeficiency virus type 1 protease (HIV-1 PR) as effective drugs against HIV/AIDS. We report here one snapshot obtained by determining the structure of enzyme-substrate complex under conditions where the catalytic activity of the enzyme is greatly reduced. The 1.76 A crystal structure shows the oligopeptide substrate, AETFYVDGAA, converted in situ into a gem-diol tetrahedral intermediate (TI). The gem-diol intermediate is neutral and one of the hydroxyl oxygens forms a very short hydrogen bond (2.2 A) with the anionic aspartate of the catalytic dyad, which is monoprotonated. Further, there is no hydrogen atom on the outer oxygen of the neutral aspartate. These two observations provide direct evidence that, in the reaction mechanism, hydrogen bonding between catalytic aspartate and scissile carbonyl oxygen facilitates water attack on the scissile carbon atom. Comparison with the structural snapshot of the biproduct complex involving the same substrate reveals the reorganization of the hydrogen bonds at the catalytic center as the enzymatic reaction progresses toward completion. Accumulation of TI in the crystals provides direct evidence that collapse of TI is the rate-limiting step of hydrolysis.

Deinococcus radiodurans has a remarkable capacity to survive exposure to extreme levels of radiation that cause hundreds of DNA double strand breaks (DSBs). DSB repair in this bacterium depends on its recombinase A protein (DrRecA). DrRecA plays a pivotal role in both extended synthesis-dependent strand annealing and slow crossover events of DSB repair during the organism's recovery from DNA damage. The mechanisms that control DrRecA activity during the D. radiodurans response to ␥ radiation exposure are unknown. Here, we show that DrRecA undergoes phosphorylation at Tyr-77 and Thr-318 by a DNA damage-responsive serine threonine/tyrosine protein kinase (RqkA). Phosphorylation modifies the activity of DrRecA in several ways, including increasing its affinity for dsDNA and its preference for dATP over ATP. Strand exchange reactions catalyzed by phosphorylated versus unphosphorylated DrRecA also differ. In silico analysis of DrRecA structure support the idea that phosphorylation can modulate crucial functions of this protein. Collectively, our findings suggest that phosphorylation of DrRecA enables the recombinase to selectively use abundant dsDNA substrate present during post-irradiation recovery for efficient DSB repair, thereby promoting the extraordinary radioresistance of D. radiodurans.Deinococcus radiodurans has a remarkable capacity to survive extreme doses of radiations and other DNA-damaging agents. Studies aimed at unraveling the molecular bases for these unusual properties have revealed that D. radiodurans encodes mechanisms for highly efficient DNA double strand break (DSB) 2 repair and oxidative stress management (1-3). DSB repair in this Gram-positive bacterium is accomplished in two phases during post-irradiation recovery (PIR); phase I is dominated by extended synthesis-dependent strand annealing (ESDSA) processes, whereas phase II involves slow crossover events in homologous recombination leading to the repair and re-establishment of the multipartite D. radiodurans genome structure (4). Despite the fact that the two phases of PIR have DNA substrates of different structures and topologies, D. radiodurans RecA (DrRecA) is required throughout DSB repair during PIR (5). Biochemical characterization of recombinant DrRecA revealed that it can form a filament on singlestranded DNA (ssDNA), exhibit co-protease activity, and utilize ATP or dATP for its energy requirements, akin to other bacterial RecA proteins (6), but it also has unusual properties. In contrast to most bacterial RecA proteins, DrRecA promotes inverse strand exchange reactions (7). Also, DrRecA promotes DNA degradation during the early phase of ESDSA repair (5), which is opposite to the function observed with Escherichia coli RecA. Transcription of DrRecA is induced in response to ␥ radiation (8, 9). However, the mechanisms by which ␥ radiation induces DrRecA expression are unusual. Inactivation of both D. radiodurans lexA genes does not attenuate ␥ radiation induction of DrRecA expression (10, 11). Thus, in contrast to many bacteria, LexA...

No abstract

The alkaline phosphatase (AP) is a bi-metalloenzyme of potential applications in biotechnology and bioremediation, in which phosphate monoesters are nonspecifically hydrolysed under alkaline conditions to yield inorganic phosphate. The hydrolysis occurs through an enzyme intermediate in which the catalytic residue is phosphorylated. The reaction, which also requires a third metal ion, is proposed to proceed through a mechanism of in-line displacement involving a trigonal bipyramidal transition state. Stabilizing the transition state by bidentate hydrogen bonding has been suggested to be the reason for conservation of an arginine residue in the active site. We report here the first crystal structure of alkaline phosphatase purified from the bacterium Sphingomonas. sp. Strain BSAR-1 (SPAP). The crystal structure reveals many differences from other APs: 1) the catalytic residue is a threonine instead of serine, 2) there is no third metal ion binding pocket, and 3) the arginine residue forming bidentate hydrogen bonding is deleted in SPAP. A lysine and an aspargine residue, recruited together for the first time into the active site, bind the substrate phosphoryl group in a manner not observed before in any other AP. These and other structural features suggest that SPAP represents a new class of APs. Because of its direct contact with the substrate phosphoryl group, the lysine residue is proposed to play a significant role in catalysis. The structure is consistent with a mechanism of in-line displacement via a trigonal bipyramidal transition state. The structure provides important insights into evolutionary relationships between members of AP superfamily.

No abstract

No abstract

Catalases are enzymes that play an important role in the detoxification of hydrogen peroxide (H 2 O 2 ) in aerobic organisms. Among catalases, haemcontaining catalases are ubiquitously distributed and their enzymatic mechanism is very well understood. On the other hand, manganese catalases that contain a bimanganese core in the active site have been less well characterized and their mode of action is not fully understood. The genome of Anabaena PCC 7120 does not show the presence of a haem catalase-like gene; instead, two ORFs encoding manganese catalases (Mn-catalases) are present. Here, the crystallization and preliminary X-ray crystallographic analysis of KatB, one of the two Mn-catalases from Anabaena, are reported. KatB was crystallized using the hanging-drop vapour-diffusion method with PEG 400 as a precipitant and calcium acetate as an additive. Diffraction data were collected in-house on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 2.2 Å resolution at 100 K. The tetragonal crystal belonged to space group P4 1 2 1 2 (or enantiomer), with unit-cell parameters a = b = 101.87, c = 138.86 Å . Preliminary X-ray diffraction analysis using the Matthews coefficient and self-rotation function suggests the presence of a trimer in the asymmetric unit.