Pathway of chymotrypsin evolution suggested by the structure of the FMN-binding protein from Desulfovibrio vulgaris (Miyazaki F)

Лиепиньш, Э. Э.; Kitamura, Masaya; Murakami, Takayuki; Nakaya, Tadao; Otting, Gottfried

doi:10.1038/nsb1297-975

Cited by 65 publications

(48 citation statements)

References 25 publications

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“…The dimeric structure has the same basic ␤-barrel scaffold used by other enzymes involved in electron transfer (33,(35)(36)(37) but displays a simpler active site in which the antibiotic and the pyruvate interact with residues from both monomers.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis of 5-Nitroimidazole Antibiotic Resistance

Leiros¹,

Kozielski-Stuhrmann²,

Kapp

et al. 2004

Journal of Biological Chemistry

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5-Nitroimidazole-based antibiotics are compounds extensively used for treating infections in humans and animals caused by several important pathogens. They are administered as prodrugs, and their activation depends upon an anaerobic 1-electron reduction of the nitro group by a reduction pathway in the cells. Bacterial resistance toward these drugs is thought to be caused by decreased drug uptake and/or an altered reduction efficiency. One class of resistant strains, identified in Bacteroides, has been shown to carry Nim genes (NimA, -B, -C, -D, and -E), which encode for reductases that convert the nitro group on the antibiotic into a non-bactericidal amine. In this paper, we have described the crystal structure of NimA from Deinococcus radiodurans (drNimA) at 1.6 Å resolution. We have shown that drNimA is a homodimer in which each monomer adopts a ␤-barrel fold. We have identified the catalytically important His-71 along with the cofactor pyruvate and antibiotic binding sites, all of which are found at the monomer-monomer interface. We have reported three additional crystal structures of drNimA, one in which the antibiotic metronidazole is bound to the protein, one with pyruvate covalently bound to His-71, and one with lactate covalently bound to His-71. Based on these structures, a reaction mechanism has been proposed in which the 2-electron reduction of the antibiotic prevents accumulation of the toxic nitro radical. This mechanism suggests that Nim proteins form a new class of reductases, conferring resistance against 5-nitroimidazole-based antibiotics.Antibiotic resistance is an increasing problem throughout the developed world, and knowledge about different resistance mechanisms is important for efficient treatment of bacterial infections. One important class of antibiotics, the 5-nitroimidazole (5-Ni) 1 drug derivatives, includes metronidazole (MTR), dimetridazole (DMZ), and tinidazole (TNZ). MTR is extensively used in the treatment of anaerobic infections caused by Trichomonas vaginalis, Entamoeba histolytica, Enterococcus species, Giardia lamblia, Clostridium species, and Bacteroides (1-4) and is also a critical ingredient of modern multidrug therapies for Helicobacter pylori eradication regimes used to control ulcers (5). The mode of action for the 5-Ni antibiotics, as illustrated in Fig. 1, has been shown to be similar in different pathogens (6 -8). The inactive prodrug enters cells by simple diffusion and is then reduced in a 1-electron reduction into the toxic compound, the short-lived radical anion R-N⅐O 2 Ϫ . This reaction is mediated by ferredoxin, which receives an electron from the pyruvate-ferredoxin oxidoreductase (PFOR) complex via conversion of pyruvate to acetyl coenzyme A (9). The resulting nitro radical anion probably causes DNA strand breaks, DNA helix destabilization, unwinding of DNA, and finally cell death (1, 2, 10, 11), and damage to other vital cell systems is also possible (6).The success of such drugs depends on the reductive activation of the nitro group on the 5-Ni drug, which ...

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Section: Discussionmentioning

confidence: 99%

Structural Basis of 5-Nitroimidazole Antibiotic Resistance

Leiros¹,

Kozielski-Stuhrmann²,

Kapp

et al. 2004

Journal of Biological Chemistry

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“…10). As found for FeR, each PheA2 subunit consists of a single domain that is organized around a six-stranded antiparallel ␤-barrel that is homologous to the FMN-binding protein from Desulfovibrio vulgaris (63). The FMN part of FAD was fit in the model structure at the same position and orientation as the FMN in FeR.…”

Section: Fig 3 Sds-page Analysis Of Protein Samples Obtained Duringmentioning

confidence: 99%

Phenol Hydroxylase from Bacillus thermoglucosidasius A7, a Two-protein Component Monooxygenase with a Dual Role for FAD

Kirchner

Westphal

Müller

et al. 2003

Journal of Biological Chemistry

125

106

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A novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55°C. PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity. The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa. PheA1 exists as a homodimer in solution and has no enzyme activity on its own. PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction. The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa. PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group. PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism. PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes. However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of shortchain flavin reductases that use FAD both as a substrate and as a prosthetic group.

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“…Three-dimensional structures of the FMN binding protein from D. Vulgaris (Miyazaki F) were determined by X-ray crystallography (Suto et al, 2000) and NMR spectroscopy (Liepinsh et al, 1997). According to these structures, Trp32 is the closest residue to Iso followed by Tyr35 and then Trp106.…”

Section: Protein Dynamics Of Fmn Binding Proteinsmentioning

confidence: 99%