Crystal structure of alkyl hydroperoxidase D like protein PA0269 from Pseudomonas aeruginosa: Homology of the AhpD-like structural family (original) (raw)
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Crystal Structure of the hypothetical protein PA4511 from Pseudomonas aeruginosa
2010
Nitroalkane oxidase (NAO) and nitronate monooxygenase (NMO) are two different types of nitroalkane oxidizing flavoenzymes identified in nature. A previous study suggested that the hypothetical protein PA4202 from Pseudomonas aeruginosa PAO1 is NMO and utilizes only anionic nitronates. However, the structural similarity between the PA4202 protein and Streptomyces ansochromogenes NAO has motivated investigation for what features of the two enzymes differentiate between the NAO and NMO activities. Herein, we report the crystal structure of PA4202 in a ternary complex with a neutral nitroethane (NE) and flavin mononucleotide (FMN) cofactor to elucidate the substrate recognition mechanism using a sitedirected mutagenesis. The ternary complex structure indicates that the NE is bound with an orientation, which is poised for the proton transfer to H183 (which is the essential first catalytic step with nitroalkanes), and subsequent reactions with FMN. Moreover, a kinetic study reveals that the catalytic reactions of the wild type and H183 mutants PA4202s with nitroalkane substrates may yield the products of hydrogen peroxide and nitrite that are specified to NAO, although they show a low catalytic efficiency. Our results provide the first structure-based molecular insight into the substrate binding property of the hypothetical protein PA4202, including the interactions with neutral nitroalkanes as the substrate.
Protoplasma, 2020
Alkyl hydroperoxide reductase subunit F (AhpF) is a well-known flavoprotein that transfers electrons from pyridine nucleotides to the peroxidase protein AhpC via redox-active disulfide centers to detoxify hydrogen peroxide. However, study of AhpF has historically been limited to particular eubacteria, and the connection between the functional and structural properties of AhpF remains unknown. The present study demonstrates the dual function of Pseudomonas aeruginosa AhpF (PaAhpF) as a reductase and a molecular chaperone. It was observed that the functions of PaAhpF are closely linked with its structural status. The reductase and foldase chaperone function of PaAhpF predominated for its low-molecular-weight (LMW) form, whereas the holdase chaperone function of PaAhpF was found associated with its high-molecular-weight (HMW) complex. Further, the present study also demonstrates the multiple function of PaAhpF in controlling oxidative and heat stresses in P. aeruginosa resistance to oxidative and heat stress.
Crystal Structure of Mycobacterium tuberculosis Catalase-Peroxidase
Journal of Biological Chemistry, 2004
The enzyme 6-hydroxymethyl-7,8-dihydropteroate synthase (DHPS) catalyzes the condensation of para-aminobenzoic acid (pABA) with 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate to form 6-hydroxymethyl-7,8dihydropteroate and pyrophosphate. DHPS is essential for the de novo synthesis of folate in prokaryotes, lower eukaryotes, and in plants, but is absent in mammals. Inhibition of this enzyme's activity by sulfonamide and sulfone drugs depletes the folate pool, resulting in growth inhibition and cell death. Here, we report the 1.7 A Ê resolution crystal structure of the binary complex of 6-hydroxymethylpterin monophosphate (PtP) with DHPS from Mycobacterium tuberculosis (Mtb), a pathogen responsible for the death of millions of human beings each year. Comparison to other DHPS structures reveals that the M. tuberculosis DHPS structure is in a unique conformation in which loop 1 closes over the active site. The Mtb DHPS structure hints at a mechanism in which both loops 1 and 2 play important roles in catalysis by shielding the active site from bulk solvent and allowing pyrophosphoryl transfer to occur. A binding mode for pABA, sulfonamides and sulfones is suggested based on: (i) the new conformation of the closed loop 1; (ii) the distribution of dapsone and sulfonamide resistance mutations; (iii) the observed direction of the bond between the 6-methyl carbon atom and the bridging oxygen atom to the a-phosphate group in the Mtb DHPS:PtP binary complex; and (iv) the conformation of loop 2 in the Escherichia coli DHPS structure. Finally, the Mtb DHPS structure reveals a highly conserved pterin binding pocket that may be exploited for the design of novel antimycobacterial agents.
A reinvestigation of the covalent structure of Pseudomonas aeruginosa cytochrome c peroxidase
FEBS Letters, 1995
The amino acid sequence of cytochrome c peroxidase from Pseudomonas aeruginosa has been determined using classical chemical degradation techniques combined with accurate mass analysis of all the generated peptides. The sequence obtained is composed of 346 amino acids and confirms the recently published cDNA-derived sequence except at one position [Ridout et al. (1995) FEBS Lett. 365, 152-154]. Based on this sequence, we propose a new model for the binding of the peroxide and the cytochrome electron donor to CCP which is in essence the reverse of the one proposed by Ellfolk et ai. [Biochim. Biophys. Acta 1080 (1991) 175-178].
Nucleotide sequence encoding the di-haem cytochrome c551 peroxidase from Pseudomonas aeruginosa
FEBS Letters, 1995
The nucleotide sequence of the gene encoding cytochrome Css ~ peroxidase from Pseudomonas aeruginosa is reported. The translated amino acid sequence differs from the sequence reported earlier by peptide mapping most significantly by the presence of a section containing an additional 20 residues. A number of minor differences are also evident. The new sequence translates to a protein containing 346 amino acids, the first 23 being typical of a hydrophobic leader peptide with a characteristic protease cleavage site. . Periplasmic proteins are normally synthesised with a hydrophobic leader peptide required for translocation, which is subsequently cleaved during maturation of the holo-protein .
Antioxidants & Redox Signaling, 2010
Bacterial antibiotic resistance is an emerging global crisis, and treatment of multidrug-resistant gram-negative infections, particularly those caused by the opportunistic human pathogen Pseudomonas aeruginosa, remains a major challenge. This problem is compounded by a lack of new antibiotics in the development pipeline: only two new classes have been developed since the 1960s, and both are indicated for multidrug-resistant gram-positive infections. A promising new approach to combat antibiotic resistance is by targeting bacterial virulence, rather than bacterial viability. The bacterial periplasmic protein DsbA represents a central point for antivirulence intervention because its oxidoreductase activity is essential for the folding and function of almost all exported virulence factors. Here we describe the three-dimensional structure of this DsbA target from P. aeruginosa, and we establish for the first time that a member of this enzyme family is capable of binding small molecules. We also describe biochemical assays that validate the redox activity of PaDsbA. Together, the structural and functional characterization of PaDsbA provides the basis for future studies aimed at designing a new class of antivirulence compounds to combat antibiotic-resistant P. aeruginosa infection. Antioxid. Redox Signal. 12, 921-931.