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The recent determination of the three-dimensional structure of urease revealed striking similarities of enzyme architecture to adenosine deaminase and phosphotriesterase, evidence of a distant evolutionary relationship that had gone undetected by one-dimensional sequence comparisons. Here, based on an analysis of conservation patterns in three dimensions, we report the discovery of the same active-site architecture in an even larger set of enzymes involved primarily in nucleotide metabolism. As a consequence, we predict the three-dimensional fold and details of the active site architecture for dihydroorotases, allantoinases, hydantoinases, AMP-, adenine and cytosine deaminases, imidazolonepropionase, aryldialkylphosphatase, chlorohydrolases, formylmethanofuran dehydrogenases, and proteins involved in animal neuronal development. Two member families are common to archaea, eubacteria, and eukaryota. Thirteen other functions supported by the same structural motif and conserved chemical mechanism apparently represent later adaptations for different substrate specificities in different cellular contexts.
Serine beta-lactamases are inhibited by phosphonate monoester monoanions. These compounds phosphonylate the active site serine hydroxyl group to form inert, covalent complexes. Since spontaneous hydrolysis of these phosphonates is generally quite slow, the beta-lactamase active site must have considerable affinity for the (presumably) pentacoordinated phosphonyl transfer transition state. Structural analogs of such a transition state might well therefore be effective and novel beta-lactamase inhibitors. Complexes of vanadate with hydroxamic acids may be able to achieve such a structure. Indeed, mixtures of these two components, but neither one alone, were found to inhibit a typical class C beta-lactamase. A Job plot of the inhibition by vanadate/benzohydroxamic acid mixtures indicated that the inhibitor was a 1:1 complex for which an inhibition constant of 4.2 microM could be calculated. A bacterial DD-peptidase, structurally similar to the beta-lactamase, was also inhibited (K(i) = 22 microM) by this complex. A similar rationale would suggest that other serine hydrolases might also be inhibited by these mixtures. In fact, chymotrypsin was inhibited by a complex of vanadate with benzohydroxamic acid (K(i) = 10 microM) and elastase by a complex with acetohydroxamic acid (K(i) = 90 microM).
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The nematode cuticle is a complex extracellular structure which is secreted by an underlying syncytium of hypodermal cells. Recent studies have demonstrated that the cuticle of parasitic nematodes is a dynamic structure with important absorptive, secretory, and enzymatic activities. In addition, the cuticle serves as a protective barrier against the host. A 48-h third stage larval Dirofilaria immitis cDNA library was immunoscreened with sera raised against larval cuticles. One clone, L3MC4 that reacted strongly with the anti-cuticle antisera was sequenced. The composite cDNA sequence comprises 2073 bp coding for a full-length protein of 590 amino acids. GenBank analysis showed that DiAsp had significant similarity to a Caenorhabditis elegans gene-product (54% identity) and to other asparaginases at the amino acid level. Escherichia coli-expressed recombinant DiAsp (rDiAsp) catalysed the hydrolysis of asparagine to aspartate and ammonia. Antibodies raised against D. immitis larval cuticles reacted with rDiAsp in immunoblots. This is the first report of identification of a cDNA clone encoding an asparaginase enzyme from a parasitic nematode.
L-aspartyl and L-asparaginyl residues in proteins spontaneously undergo intra-residue rearrangements forming isoaspartyl/beta-aspartyl residues linked through their side-chain beta-carboxyl group with the following amino acid. In order to avoid accumulation of isoaspartyl dipeptides left over from protein degradation, some bacteria have developed specialized isoaspartyl/beta-aspartyl zinc dipeptidases sequentially unrelated to other peptidases, which also poorly degrade alpha-aspartyl dipeptides. We have expressed and crystallized the 390 amino acid residue isoaspartyl dipeptidase (IadA) from E.coli, and have determined its crystal structure in the absence and presence of the phosphinic inhibitor Asp-Psi[PO(2)CH(2)]-LeuOH. This structure reveals an octameric particle of 422 symmetry, with each polypeptide chain organized in a (alphabeta)(8) TIM-like barrel catalytic domain attached to a U-shaped beta-sandwich domain. At the C termini of the beta-strands of the beta-barrel, the two catalytic zinc ions are surrounded by four His, a bridging carbamylated Lys and an Asp residue, which seems to act as a proton shuttle. A large beta-hairpin loop protruding from the (alphabeta)(8) barrel is disordered in the free peptidase, but forms a flap that stoppers the barrel entrance to the active center upon binding of the dipeptide mimic. This isoaspartyl dipeptidase shows strong topological homology with the alpha-subunit of the binickel-containing ureases, the dinuclear zinc dihydroorotases, hydantoinases and phosphotriesterases, and the mononuclear adenosine and cytosine deaminases, which all are catalyzing hydrolytic reactions at carbon or phosphorous centers. Thus, nature has adapted an existing fold with catalytic tools suitable for hydrolysis of amide bonds to the binding requirements of a peptidase.
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Auxin autonomous growth of most crown gall tumor cells requires the expression of two auxin biosynthesizing genes (tms 1 and tms 2) from the T-DNA of Agrobacterium tumefaciens. The potential role of the tms 2 locus to affect auxin accumulation was studied by measuring the activity of its gene product, indoleacetamide hydrolase (AH), in cloned cells of tobacco (Nicotiana tabacum) transformed by the A6 strain of Agrobacterium tumefaciens. AH activity followed a consistent pattern over a 30 day culture cycle with a peak at 10 to 14 days. This same pattern was observed in a number of independently isolated clones as well as in uncioned tumor tissue, suggesting that AH activation is a regular process in wounded, transformed cells. Transfer of unwounded tissue to fresh media resulted in a similar pattern of AH activation, but with the peak activity only about 50 percent of the cut tissues. These results show that the tms 2 encoded AH activity is modulated over the culture cycle, and that the modulation is affected by wounding and supplying fresh nutrients in the medium. AH activity correlated closely with free indoleacetic acid levels which suggests that it can be an important determinant in controlling free IAA levels in transformed cells.
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