Chengfu Xu

Enzymatic neutralization of the chemical warfare agent VX: evolution of phosphotriesterase for phosphorothiolate hydrolysis.

The V-type nerve agents (VX and VR) are among the most toxic substances known. The high toxicity and environmental persistence of VX make the development of novel decontamination methods particularly important. The enzyme phosphotriesterase (PTE) is capable of hydrolyzing VX but with an enzymatic efficiency more than 5 orders of magnitude lower than with its best substrate, paraoxon. PTE has previously proven amenable to directed evolution for the improvement of catalytic activity against selected compounds through the manipulation of active-site residues. Here, a series of sequential two-site mutational libraries encompassing 12 active-site residues of PTE was created. The libraries were screened for catalytic activity against a new VX analogue, DEVX, which contains the same thiolate leaving group of VX coupled to a diethoxyphosphate core rather than the ethoxymethylphosphonate core of VX. The evolved catalytic activity with DEVX was enhanced 26-fold relative to wild-type PTE. Further improvements were facilitated by targeted error-prone PCR mutagenesis of loop-7, and additional PTE variants were identified with up to a 78-fold increase in the rate of DEVX hydrolysis. The best mutant hydrolyzed the racemic nerve agent VX with a value of kcat/Km = 7 × 10(4) M(-1) s(-1), a 230-fold improvement relative to wild-type PTE. The highest turnover number achieved by the mutants created for this investigation was 137 s(-1), an enhancement of 152-fold relative to wild-type PTE. The stereoselectivity for the hydrolysis of the two enantiomers of VX was relatively low. These engineered mutants of PTE are the best catalysts ever reported for the hydrolysis of nerve agent VX.

Functional Identification of Incorrectly Annotated Prolidases from the Amidohydrolase Superfamily of Enzymes

The substrate profiles for two proteins from Caulobacter crescentus CB15 (Cc2672 and Cc3125) and one protein (Sgx9359b) derived from a DNA sequence ( gi|44368820 ) isolated from the Sargasso Sea were determined using combinatorial libraries of dipeptides and N-acyl derivatives of amino acids. These proteins are members of the amidohydrolase superfamily and are currently misannotated in NCBI as catalyzing the hydrolysis of l-Xaa-l-Pro dipeptides. Cc2672 was shown to catalyze the hydrolysis of l-Xaa-l-Arg/Lys dipeptides and the N-acetyl and N-formyl derivatives of lysine and arginine. This enzyme will also hydrolyze longer peptides that terminate in either lysine or arginine. The N-methyl phosphonate derivative of l-lysine was a potent competitive inhibitor of Cc2672 with a K(i) value of 120 nM. Cc3125 was shown to catalyze the hydrolysis of l-Xaa-l-Arg/Lys dipeptides but will not hydrolyze tripeptides or the N-formyl and N-acetyl derivatives of lysine or arginine. The substrate profile for Sgx9359b is similar to that of Cc2672 except that compounds with a C-terminal lysine are not recognized as substrates. The X-ray structure of Sgx9359b was determined to a resolution of 2.3 A. The protein folds as a (beta/alpha)(8)-barrel and self-associates to form a homooctamer. The active site is composed of a binuclear metal center similar to that found in phosphotriesterase and dihydroorotase. In one crystal form, arginine was bound adventitiously to the eight active sites within the octamer. The orientation of the arginine in the active site identified the structural determinants for recognition of the alpha-carboxylate and the positively charged side chains of arginine-containing substrates. This information was used to identify 18 other bacterial sequences that possess identical or similar substrate profiles.

Structure and Catalytic Mechanism of LigI: Insight into the Amidohydrolase Enzymes of cog3618 and Lignin Degradation

LigI from Sphingomonas paucimobilis catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate and 4-carboxy-2-hydroxymuconate in the degradation of lignin. This protein is a member of the amidohydrolase superfamily of enzymes. The protein was expressed in Escherichia coli and then purified to homogeneity. The purified recombinant enzyme does not contain bound metal ions, and the addition of metal chelators or divalent metal ions to the assay mixtures does not affect the rate of product formation. This is the first enzyme from the amidohydrolase superfamily that does not require a divalent metal ion for catalytic activity. The kinetic constants for the hydrolysis of PDC are 340 s(-1) and 9.8 × 10(6) M(-1) s(-1) (k(cat) and k(cat)/K(m), respectively). The pH dependence on the kinetic constants suggests that a single active site residue must be deprotonated for the hydrolysis of PDC. The site of nucleophilic attack was determined by conducting the hydrolysis of PDC in (18)O-labeled water and subsequent (13)C nuclear magnetic resonance analysis. The crystal structures of wild-type LigI and the D248A mutant in the presence of the reaction product were determined to a resolution of 1.9 Å. The C-8 and C-11 carboxylate groups of PDC are coordinated within the active site via ion pair interactions with Arg-130 and Arg-124, respectively. The hydrolytic water molecule is activated by the transfer of a proton to Asp-248. The carbonyl group of the lactone substrate is activated by electrostatic interactions with His-180, His-31, and His-33.

Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase

Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K(i) of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pK(a) of 6.0, and Zn-CDA has a kinetic pK(a) of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k(cat) and k(cat)/K(m), consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.

Structure-Based Function Discovery of an Enzyme for the Hydrolysis of Phosphorylated Sugar Lactones

Two enzymes of unknown function from the cog1735 subset of the amidohydrolase superfamily (AHS), LMOf2365_2620 (Lmo2620) from Listeria monocytogenes str. 4b F2365 and Bh0225 from Bacillus halodurans C-125, were cloned, expressed, and purified to homogeneity. The catalytic functions of these two enzymes were interrogated by an integrated strategy encompassing bioinformatics, computational docking to three-dimensional crystal structures, and library screening. The three-dimensional structure of Lmo2620 was determined at a resolution of 1.6 Å with two phosphates and a binuclear zinc center in the active site. The proximal phosphate bridges the binuclear metal center and is 7.1 Å from the distal phosphate. The distal phosphate hydrogen bonds with Lys-242, Lys-244, Arg-275, and Tyr-278. Enzymes within cog1735 of the AHS have previously been shown to catalyze the hydrolysis of substituted lactones. Computational docking of the high-energy intermediate form of the KEGG database to the three-dimensional structure of Lmo2620 highly enriched anionic lactones versus other candidate substrates. The active site structure and the computational docking results suggested that probable substrates would likely include phosphorylated sugar lactones. A small library of diacid sugar lactones and phosphorylated sugar lactones was synthesized and tested for substrate activity with Lmo2620 and Bh0225. Two substrates were identified for these enzymes, D-lyxono-1,4-lactone-5-phosphate and l-ribono-1,4-lactone-5-phosphate. The k(cat)/K(m) values for the cobalt-substituted enzymes with these substrates are ~10(5) M(-1) s(-1).

Functional identification and structure determination of two novel prolidases from cog1228 in the amidohydrolase superfamily .

Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 were cloned, expressed, and purified to homogeneity. The two proteins, Sgx9260c ( gi|44242006 ) and Sgx9260b ( gi|44479596 ), were derived from environmental DNA samples originating from the Sargasso Sea. The catalytic function and substrate profiles for Sgx9260c and Sgx9260b were determined using a comprehensive library of dipeptides and N-acyl derivative of l-amino acids. Sgx9260c catalyzes the hydrolysis of Gly-l-Pro, l-Ala-l-Pro, and N-acyl derivatives of l-Pro. The best substrate identified to date is N-acetyl-l-Pro with a value of k(cat)/K(m) of 3 x 10(5) M(-1) s(-1). Sgx9260b catalyzes the hydrolysis of l-hydrophobic l-Pro dipeptides and N-acyl derivatives of l-Pro. The best substrate identified to date is N-propionyl-l-Pro with a value of k(cat)/K(m) of 1 x 10(5) M(-1) s(-1). Three-dimensional structures of both proteins were determined by X-ray diffraction methods (PDB codes 3MKV and 3FEQ ). These proteins fold as distorted (beta/alpha)(8)-barrels with two divalent cations in the active site. The structure of Sgx9260c was also determined as a complex with the N-methylphosphonate derivative of l-Pro (PDB code 3N2C ). In this structure the phosphonate moiety bridges the binuclear metal center, and one oxygen atom interacts with His-140. The alpha-carboxylate of the inhibitor interacts with Tyr-231. The proline side chain occupies a small substrate binding cavity formed by residues contributed from the loop that follows beta-strand 7 within the (beta/alpha)(8)-barrel. A total of 38 other proteins from cog1228 are predicted to have the same substrate profile based on conservation of the substrate binding residues. The structure of an evolutionarily related protein, Cc2672 from Caulobacter crecentus, was determined as a complex with the N-methylphosphonate derivative of l-arginine (PDB code 3MTW ).

Annotating enzymes of uncertain function: the deacylation of D-amino acids by members of the amidohydrolase superfamily.

The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k(cat)/K(m) = 5.8 x 10(6) M(-1) s(-1)), N-acetyl-d-glutamate (k(cat)/K(m) = 5.2 x 10(6) M(-1) s(-1)), and l-methionine-d-glutamate (k(cat)/K(m) = 3.4 x 10(5) M(-1) s(-1)). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k(cat)/K(m) = 3.2 x 10(4) M(-1) s(-1)), N-acetyl-d-tryptophan (k(cat)/K(m) = 4.1 x 10(4) M(-1) s(-1)), and l-tyrosine-d-leucine (k(cat)/K(m) = 1.5 x 10(4) M(-1) s(-1)). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the alpha-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional approximately 250 sequences identified as members of this group suggest that there are no simple motifs that allow prediction of substrate specificity for most of these unknowns, highlighting the challenges for computational annotation of some groups of homologous proteins.

Functional annotation of two new carboxypeptidases from the amidohydrolase superfamily of enzymes.

Two proteins from the amidohydrolase superfamily of enzymes were cloned, expressed, and purified to homogeneity. The first protein, Cc0300, was from Caulobacter crescentus CB-15 (Cc0300), while the second one (Sgx9355e) was derived from an environmental DNA sequence originally isolated from the Sargasso Sea ( gi|44371129 ). The catalytic functions and the substrate profiles for the two enzymes were determined with the aid of combinatorial dipeptide libraries. Both enzymes were shown to catalyze the hydrolysis of l-Xaa-l-Xaa dipeptides in which the amino acid at the N-terminus was relatively unimportant. These enzymes were specific for hydrophobic amino acids at the C-terminus. With Cc0300, substrates terminating in isoleucine, leucine, phenylalanine, tyrosine, valine, methionine, and tryptophan were hydrolyzed. The same specificity was observed with Sgx9355e, but this protein was also able to hydrolyze peptides terminating in threonine. Both enzymes were able to hydrolyze N-acetyl and N-formyl derivatives of the hydrophobic amino acids and tripeptides. The best substrates identified for Cc0300 were l-Ala-l-Leu with k(cat) and k(cat)/K(m) values of 37 s(-1) and 1.1 x 10(5) M(-1) s(-1), respectively, and N-formyl-l-Tyr with k(cat) and k(cat)/K(m) values of 33 s(-1) and 3.9 x 10(5) M(-1) s(-1), respectively. The best substrate identified for Sgx9355e was l-Ala-l-Phe with k(cat) and k(cat)/K(m) values of 0.41 s(-1) and 5.8 x 10(3) M(-1) s(-1). The three-dimensional structure of Sgx9355e was determined to a resolution of 2.33 A with l-methionine bound in the active site. The alpha-carboxylate of the methionine is ion-paired to His-237 and also hydrogen bonded to the backbone amide groups of Val-201 and Leu-202. The alpha-amino group of the bound methionine interacts with Asp-328. The structural determinants for substrate recognition were identified and compared with other enzymes in this superfamily that hydrolyze dipeptides with different specificities.

Structure, Mechanism, and Substrate Profile for Sco3058: The Closest Bacterial Homologue to Human Renal Dipeptidase

Human renal dipeptidase, an enzyme associated with glutathione metabolism and the hydrolysis of beta-lactams, is similar in sequence to a cluster of approximately 400 microbial proteins currently annotated as nonspecific dipeptidases within the amidohydrolase superfamily. The closest homologue to the human renal dipeptidase from a fully sequenced microbe is Sco3058 from Streptomyces coelicolor. Dipeptide substrates of Sco3058 were identified by screening a comprehensive series of l-Xaa-l-Xaa, l-Xaa-d-Xaa, and d-Xaa-l-Xaa dipeptide libraries. The substrate specificity profile shows that Sco3058 hydrolyzes a broad range of dipeptides with a marked preference for an l-amino acid at the N-terminus and a d-amino acid at the C-terminus. The best substrate identified was l-Arg-d-Asp (k(cat)/K(m) = 7.6 x 10(5) M(-1) s(-1)). The three-dimensional structure of Sco3058 was determined in the absence and presence of the inhibitors citrate and a phosphinate mimic of l-Ala-d-Asp. The enzyme folds as a (beta/alpha)(8) barrel, and two zinc ions are bound in the active site. Site-directed mutagenesis was used to probe the importance of specific residues that have direct interactions with the substrate analogues in the active site (Asp-22, His-150, Arg-223, and Asp-320). The solvent viscosity and kinetic effects of D(2)O indicate that substrate binding is relatively sticky and that proton transfers do not occurr during the rate-limiting step. A bell-shaped pH-rate profile for k(cat) and k(cat)/K(m) indicated that one group needs to be deprotonated and a second group must be protonated for optimal turnover. Computational docking of high-energy intermediate forms of l/d-Ala-l/d-Ala to the three-dimensional structure of Sco3058 identified the structural determinants for the stereochemical preferences for substrate binding and turnover.

Prospecting for Unannotated Enzymes: Discovery of a 3',5'-Nucleotide Bisphosphate Phosphatase within the Amidohydrolase Superfamily.

In bacteria, 3′,5′-adenosine bisphosphate (pAp) is generated from 3′-phosphoadenosine 5′-phosphosulfate in the sulfate assimilation pathway, and from coenzyme A by the transfer of the phosphopantetheine group to the acyl-carrier protein. pAp is subsequently hydrolyzed to 5′-AMP and orthophosphate, and this reaction has been shown to be important for superoxide stress tolerance. Herein, we report the discovery of the first instance of an enzyme from the amidohydrolase superfamily that is capable of hydrolyzing pAp. Crystal structures of Cv1693 from Chromobacterium violaceum have been determined to a resolution of 1.9 Å with AMP and orthophosphate bound in the active site. The enzyme has a trinuclear metal center in the active site with three Mn2+ ions. This enzyme (Cv1693) belongs to the Cluster of Orthologous Groups cog0613 from the polymerase and histidinol phosphatase family of enzymes. The values of kcat and kcat/Km for the hydrolysis of pAp are 22 s–1 and 1.4 × 106 M–1 s–1, respectively. The enzyme is promiscuous and is able to hydrolyze other 3′,5′-bisphosphonucleotides (pGp, pCp, pUp, and pIp) and 2′-deoxynucleotides with comparable catalytic efficiency. The enzyme is capable of hydrolyzing short oligonucleotides (pdA)5, albeit at rates much lower than that of pAp. Enzymes from two other enzyme families have previously been found to hydrolyze pAp at physiologically significant rates. These enzymes include CysQ from Escherichia coli (cog1218) and YtqI/NrnA from Bacillus subtilis (cog0618). Identification of the functional homologues to the experimentally verified pAp phosphatases from cog0613, cog1218, and cog0618 suggests that there is relatively little overlap of enzymes with this function in sequenced bacterial genomes.