X-Ray Structure and Mutagenesis Studies of the N-Isopropylammelide Isopropylaminohydrolase, AtzC.

The N-isopropylammelide isopropylaminohydrolase from Pseudomonas sp. strain ADP, AtzC, provides the third hydrolytic step in the mineralization of s-triazine herbicides, such as atrazine. We obtained the X-ray crystal structure of AtzC at 1.84 Å with a weak inhibitor bound in the active site and then used a combination of in silico docking and site-directed mutagenesis to understand the interactions between AtzC and its substrate, isopropylammelide. The substitution of an active site histidine residue (His249) for an alanine abolished the enzyme's catalytic activity. We propose a plausible catalytic mechanism, consistent with the biochemical and crystallographic data obtained that is similar to that found in carbonic anhydrase and other members of subtype III of the amidohydrolase family.

X-ray structure of the amidase domain of AtzF, the allophanate hydrolase from the cyanuric acid-mineralizing multienzyme complex.

The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis, when purified enzyme was tested in vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product.

Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP.

The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Šresolution and adopted space group P21, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, ß = 106.6°.

Cyanuric acid hydrolase: evolutionary innovation by structural concatenation.

The cyanuric acid hydrolase, AtzD, is the founding member of a newly identified family of ring-opening amidases. We report the first X-ray structure for this family, which is a novel fold (termed the 'Toblerone' fold) that likely evolved via the concatenation of monomers of the trimeric YjgF superfamily and the acquisition of a metal binding site. Structures of AtzD with bound substrate (cyanuric acid) and inhibitors (phosphate, barbituric acid and melamine), along with mutagenesis studies, allowed the identification of the active site. The AtzD monomer, active site and substrate all possess threefold rotational symmetry, to the extent that the active site possesses three potential Ser-Lys catalytic dyads. A single catalytic dyad (Ser85-Lys42) is hypothesized, based on biochemical evidence and crystallographic data. A plausible catalytic mechanism based on these observations is also presented. A comparison with a homology model of the related barbiturase, Bar, was used to infer the active-site residues responsible for substrate specificity, and the phylogeny of the 68 AtzD-like enzymes in the database were analysed in light of this structure-function relationship.

Identification and characterization of two families of F420 H2-dependent reductases from Mycobacteria that catalyse aflatoxin degradation.

Aflatoxins are polyaromatic mycotoxins that contaminate a range of food crops as a result of fungal growth and contribute to serious health problems in the developing world because of their toxicity and mutagenicity. Although relatively resistant to biotic degradation, aflatoxins can be metabolized by certain species of Actinomycetales. However, the enzymatic basis for their breakdown has not been reported until now. We have identified nine Mycobacterium smegmatis enzymes that utilize the deazaflavin cofactor F(420) H(2) to catalyse the reduction of the α,ß-unsaturated ester moiety of aflatoxins, activating the molecules for spontaneous hydrolysis and detoxification. These enzymes belong to two previously uncharacterized F(420) H(2) dependent reductase (FDR-A and -B) families that are distantly related to the flavin mononucleotide (FMN) dependent pyridoxamine 5'-phosphate oxidases (PNPOxs). We have solved crystal structures of an enzyme from each FDR family and show that they, like the PNPOxs, adopt a split barrel protein fold, although the FDRs also possess an extended and highly charged F(420) H(2) binding groove. A general role for these enzymes in xenobiotic metabolism is discussed, including the observation that the nitro-reductase Rv3547 from Mycobacterium tuberculosis that is responsible for the activation of bicyclic nitroimidazole prodrugs belongs to the FDR-A family.

Defective importin beta recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations.

The architectural transcription factor SRY (sex-determining region of the Y chromosome) plays a key role in sex determination as indicated by the fact that mutations in SRY are responsible for XY gonadal dysgenesis in humans. Although many SRY mutations reduce DNA-binding/bending activity, it is not clear how SRY mutations that do not affect interaction with DNA contribute to disease. The SRY high-mobility group domain harbors two nuclear localization signals (NLSs), and here we examine SRY from four XY females with missense mutations in these signals. In all cases, mutant SRY protein is partly localized to the cytoplasm, whereas wild-type SRY is strictly nuclear. Each NLS can independently direct nuclear transport of a carrier protein in vitro and in vivo, with mutations in either affecting the rate and extent of nuclear accumulation. The N-terminal NLS function is independent of the conventional NLS-binding importins (IMPs) and requires unidentified cytoplasmic transport factors, whereas the C-terminal NLS is recognized by IMPbeta. The SRY-R133W mutant shows reduced IMPbeta binding as a direct consequence of the sex-reversing C-terminal NLS mutation. Of the N-terminal NLS mutants examined, SRY-R62G unexpectedly shows a marked reduction in IMPbeta binding, whereas SRY-R75N and SRY-R76P show normal IMPbeta binding, suggesting defects in the IMP-independent pathway. We conclude that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination. This study documents cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate in the nucleus.