The outer membrane protein A (OmpA) plays important roles in anchoring of the outer membrane to the bacterial cell wall. The C-terminal periplasmic domain of OmpA (OmpA-like domain) associates with the peptidoglycan (PGN) layer noncovalently. However, there is a paucity of information on the structural aspects of the mechanism of PGN recognition by OmpA-like domains. To elucidate this molecular recognition process, we solved the high-resolution crystal structure of an OmpA-like domain from Acinetobacter baumannii bound to diaminopimelate (DAP), a unique bacterial amino acid from the PGN. The structure clearly illustrates that two absolutely conserved Asp271 and Arg286 residues are the key to the binding to DAP of PGN. Identification of DAP as the central anchoring site of PGN to OmpA is further supported by isothermal titration calorimetry and a pulldown assay with PGN. An NMR-based computational model for complexation between the PGN and OmpA emerged, and this model is validated by determining the crystal structure in complex with a synthetic PGN fragment. These structural data provide a detailed glimpse of how the anchoring of OmpA to the cell wall of gram-negative bacteria takes place in a DAP-dependent manner.

IsdG and IsdI are paralogous proteins that are intracellular components of a complex heme uptake system in Staphylococcus aureus. IsdG and IsdI were shown previously to reductively degrade hemin. Crystal structures of the apoproteins show that these proteins belong to a newly identified heme degradation family distinct from canonical eukaryotic and prokaryotic heme oxygenases. Here we report the crystal structures of an inactive N7A variant of IsdG in complex with Fe 3؉ -protoporphyrin IX (IsdG-hemin) and of IsdI in complex with cobalt protoporphyrin IX (IsdI-CoPPIX) to 1.8 Å or better resolution. These structures show that the metalloporphyrins are buried into similar deep clefts such that the propionic acids form salt bridges to two Arg residues. His 77 (IsdG) or His 76 (IsdI), a critical residue required for activity, is coordinated to the Fe 3؉ or Co 3؉ atoms, respectively. The bound porphyrin rings form extensive steric interactions in the binding cleft such that the rings are highly distorted from the plane. This distortion is best described as ruffled and places the ␤-and ␦-meso carbons proximal to the distal oxygen-binding site. In the IsdG-hemin structure, Fe 3؉ is pentacoordinate, and the distal side is occluded by the side chain of Ile 55 . However, in the structure of IsdI-CoPPIX, the distal side of the CoPPIX accommodates a chloride ion in a cavity formed through a conformational change in Ile 55 . The chloride ion participates in a hydrogen bond to the side chain amide of Asn 6 . Together the structures suggest a reaction mechanism in which a reactive peroxide intermediate proceeds with nucleophilic oxidation at the ␤-or ␦-meso carbon of the hemin.Staphylococcus aureus is a leading cause of hospital-acquired bacterial infections (1). The establishment of methicillin-resistant strains of S. aureus is a concern in both the clinic and, more recently, within the community (2, 3). Iron uptake pathways have received significant attention because of the requirement of iron for the growth of most organisms (4). For human pathogens, iron concentrations are limited by host storage, transport, and innate immune mechanisms (2, 5). Many bacterial pathogens have sophisticated systems to directly utilize host iron sources to satisfy their physiological requirements. Heme-iron represents the most abundant iron source in the human body, accounting for ϳ75% of the total iron (6). This heme-iron is predominantly found within hemoglobin in circulating red blood cells and myoglobin of muscle cells. Because of its abundance, an ability to acquire heme-iron from host sources represents a significant advantage for bacterial pathogens (7-9).S. aureus acquires heme-iron predominantly through the Isd (iron-regulated surface determinant) system. IsdA, IsdB, IsdC, and IsdH/HarA are cell wall-anchored proteins (10) that contain heme-binding NEAT domains (11, 12). The host hemoprotein hemoglobin and its carrier protein haptoglobin are bound by IsdB and IsdH at the cell surface. Heme is proposed to be removed from hemoglobin and ...

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The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-Å resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an ␣ ؉ ␤-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. Nitroaromatic compounds including nitrofurans, nitropyrenes, and nitrobenzenes have been used as antimicrobial agents, food additives and raw materials in several industrial processes (1-5), and as a result are distributed widely around the environment. Many of these compounds are toxic, mutagenic, or carcinogenic (6 -8). It is believed that enzymatic transformation is needed for nitroaromatic compounds to show these serious effects (9, 10). The reduction of a nitro group of a parent nitrocompound is a key step of this process (11,12). Enzymes, which catalyze the reduction of nitrocompounds using a reduced pyridine nucleotide, are termed nitroreductases and are distinguished by their sensitivity of activity to oxygen (9, 10).The oxygen-sensitive enzymes can catalyze nitroreduction only under anaerobic conditions. A nitro-anion radical formed by a one-electron transfer is immediately reoxidized in the presence of oxygen to a parent nitrocompound and superoxide (13,14). In this futile cycle, reducing equivalents are consumed without the progress of nitroreduction and nitrocompounds perform as a catalyst to reduce oxygen. On the other hand, the oxygen-insensitive enzymes catalyze an obligatory two-electron reduction. A nitro group of a parent nitrocompound is reduced by a series of two-electron transfers, through nitroso and hydroxylamine intermediates, and finally to an amino group (13). The hydroxylamine intermediate arising from the four-electron transfer in total is found to be toxic, carcinogenic, or mutagenic.Three proteins with oxygen-insensitive nitroreductase activity in Escherichia coli have been identified (15). NfsA 1 is the major component, while NfsB and NfsC are minor components. NfsA and NfsB have been well studied relative to NfsC. NfsA and NfsB have similar enzymatic property, although NfsA has only 7% identity with NfsB on the amino acid sequence alignment. Both NfsA and NfsB are flavoenzymes with FMN as the prosthetic group and catalyze the reduction of nitrocompounds by Ping Pong Bi Bi kinetics (16,17). Counterparts of NfsA and NfsB, found in luminescent bacteria (16,17), are flavin reductase (FRP) of Vibrio harveyi (18) and flavin reductase (FRase I) of Vibrio fischeri (19), respectively. Enzyme that resembles FRP in the amino acid sequence alignment is also found in Bacillus subtilus and is called NfrA1 (20). A comparison of * This work was supported in part by grants-in-aid for Scientific Res...

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