Burkholderia genome analysis reveals new enzymes belonging to the nitrilase superfamily. The amidase of Burkholderia cepacia (hospital isolate)

Burkholderia cepacia (formerly Pseudomonas cepacia) grows in media containing acetamide or propionamide as C and N sources. Chromosomal DNA from a hospital isolate of B. cepacia served as a template in PCRs using primers designed for the amplification of the P. aeruginosa amiE gene that encodes an aliphatic amidase. Partial sequencing of the PCR products gave a translated sequence 100% identical with the amino acid sequence of P. aeruginosa amidase. A search of Burkholderia genomes detected a putative amidase in B. cepacia J2315 with high identity to the P. aeruginosa amidase and predicted that other Burkholderia species also possessed CN_hydrolases that use the same catalytic triad (Glu–Lys–Cys) as amidase. Superimposition of theoretical three-dimensional models suggested that differences in the amino acid sequences between amidases from B. cepacia (hospital isolate) and B. cepacia J2315 do not affect their three-dimensional structure.     

Cloning, sequence analysis and expression of the gene encoding a novel wide-spectrum amidase belonging to the amidase signature superfamily from Achromobacter xylosoxidans

Amidases are very important enzymes for industrial biocatalysis. We scored a novel amidase by screening the Achromobacter xylosoxidans gene library with cephalosporin analogous amides. The gene coding for the enzyme, designated ana, was cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis of ana showed it to be an amidase signature family member. Interestingly, we noted that almost all Ana homologous amidases are from human pathogens responsible for chronic lung infections. Knowing the genetic context of Ana and its homologous amidases, we suggest that they could be a part of transposon structure. Ana can efficiently hydrolyze a series of cephalosporin analogous amides, including amides with an aninine, p-nitro-aninine, and beta-naphthylamine moiety, while cephalosporin could not serve as its substrate.     

Crystal structures of biotin protein ligase from Pyrococcus horikoshii OT3 and its complexes: structural basis of biotin activation

Biotin protein ligase (EC 6.3.4.15) catalyses the synthesis of an activated form of biotin, biotinyl-5'-AMP, from substrates biotin and ATP followed by biotinylation of the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase. The three-dimensional structure of biotin protein ligase from Pyrococcus horikoshii OT3 has been determined by X-ray diffraction at 1.6A resolution. The structure reveals a homodimer as the functional unit. Each subunit contains two domains, a larger N-terminal catalytic domain and a smaller C-terminal domain. The structural feature of the active site has been studied by determination of the crystal structures of complexes of the enzyme with biotin, ADP and the reaction intermediate biotinyl-5'-AMP at atomic resolution. This is the first report of the liganded structures of biotin protein ligase with nucleotide and biotinyl-5'-AMP. The structures of the unliganded and the liganded forms are isomorphous except for an ordering of the active site loop upon ligand binding. Catalytic binding sites are suitably arranged to minimize the conformational changes required during the reaction, as the pockets for biotin and nucleotide are located spatially adjacent to each other in a cleft of the catalytic domain and the pocket for biotinyl-5'-AMP binding mimics the combination of those of the substrates. The exact locations of the ligands and the active site residues allow us to propose a general scheme for the first step of the reaction carried out by biotin protein ligase in which the positively charged epsilon-amino group of Lys111 facilitates the nucleophilic attack on the ATP alpha-phosphate group by the biotin carboxyl oxygen atom and stabilizes the negatively charged intermediates.     

A peptidoglycan recognition protein from Sciaenops ocellatus is a zinc amidase and a bactericide with a substrate range limited to Gram-positive bacteria

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46-94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn²⁺-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection.     

A novel esterase from a psychrotrophic bacterium, Acinetobacter sp. strain no. 6, that belongs to the amidase signature family

A novel esterase that belongs to the amidase signature family was found in a psychrotrophic bacterium, Acinetobacter sp. strain no. 6, isolated from Siberian soil. The gene coding for the esterase, named EstA8, was cloned, and an open reading frame of 1488 bp corresponding to 496 amino acid residues was identified. EstA8 showed 30% sequence identity with 6-aminohexanoate-cyclic-dimer hydrolases from Pseudomonas sp. strain NK87 and Flavobacterium sp. strain K172, which degrade a by-product of the nylon-6 industry. EstA8 was overproduced in Escherichia coli JM109 under the control of the lac promoter of pUC118 and purified. Consistent with the fact that the source microorganism is cold-adapted, the enzyme was unstable at moderate temperatures. It lost 75% of its original activity by incubation at 40 °C for 30 min. Despite its structural similarity to 6-aminohexanoate-cyclic-dimer hydrolase, 6-aminohexanoate cyclic dimer did not serve as the substrate. EstA8 is a member of the amidase signature family, but its esterase activity toward p-nitrophenyl esters, such as p-nitrophenyl acetate, was much higher than its amidase activity toward p-nitroanilides, such as p-nitroacetanilide.     

The contribution of peripheral stem-loops to the catalytic activity of archaeal RNase P RNA from Pyrococcus horikoshii OT3

We investigated the contribution of peripheral stem-loops to the catalytic activity of an archaeal RNase P RNA, PhopRNA, from Pyrococcus horikoshii OT3. PhopRNA mutants, in which the stem-loops were individually deleted, were prepared and characterized with respect to precursor tRNA (pre-tRNA) cleavage activity in the presence of five RNase P proteins. All the mutants retained the activity to some extent, indicating that they are moderately implicated in catalysis. Further characterization suggested that the stem-loops serve largely as binding sites for the proteins, and that their interactions are predominantly involved in stabilization of the active conformation of PhopRNA.     

The Contribution of Peripheral Stem-Loops to the Catalytic Activity of Archaeal RNase P RNA from Pyrococcus horikoshii OT3

We investigated the contribution of peripheral stem-loops to the catalytic activity of an archaeal RNase P RNA, PhopRNA, from Pyrococcus horikoshii OT3. PhopRNA mutants, in which the stem-loops were individually deleted, were prepared and characterized with respect to precursor tRNA (pre-tRNA) cleavage activity in the presence of five RNase P proteins. All the mutants retained the activity to some extent, indicating that they are moderately implicated in catalysis. Further characterization suggested that the stem-loops serve largely as binding sites for the proteins, and that their interactions are predominantly involved in stabilization of the active conformation of PhopRNA.     

Alteration of metal ions improves the activity and thermostability of aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

Recombinant l-aminoacylase (PhoACY) from a hyperthermophilic archeon, Pyrococcus horikoshii, is a zinc-containing metalloenzyme. When the zinc was substituted by Mn²⁺ or Ni²⁺, its specific activity was significantly increased with acetyl-l-methionine as a substrate. The thermostability of PhoACY was improved when it was incubated with 1 mM Zn²⁺, Mn²⁺ or Ni²⁺. The enzyme with external Zn²⁺ addition had no significant loss of the activity when held at 90°C for up to 12 h and moreover had more than a 10-fold longer half-life even at 100°C, compared to the enzyme without Zn²⁺ addition. A thermostable structure of the enzyme associated with zinc binding is described based on differential scanning calorimetry.     

极端嗜热古菌Pyrococcus horikoshii几丁二糖脱乙酰酶的克隆、表达及性质研究

利用PCR扩增技术从极端嗜热古菌Pyrococcus horikoshii中得到预测为几丁二糖脱乙酰酶的基因(Dacph,PH0499),将其克隆入表达质粒pET15b,并在E.coliBL21_codonPlus(DE3)_RIL中表达获得可溶的Dacph重组蛋白(31.6kDa),TLC分析证明Dacph能够脱去N_乙酰氨基葡萄糖及几丁二糖的一个乙酰基,并与氨基葡萄糖苷酶(BglAPh)共同作用水解几丁二糖生成氨基葡萄糖,从而被命名为一种几丁二糖脱乙酰酶。与Pyrococcus horikoshii中外切氨基葡萄糖苷酶等共同作用,Dacph可能在嗜热球古菌独特的几丁质降解途径中起重要作用。     

Analyzing the binding of Co(II)-specific inhibitors to the methionyl aminopeptidases from Escherichia coli and Pyrococcus furiosus

Methionine aminopeptidases (MetAPs) represent a unique class of protease that is capable of the hydrolytic removal of an N-terminal methionine residue from nascent polypeptide chains. MetAPs are physiologically important enzymes; hence, there is considerable interest in developing inhibitors that can be used as antiangiogenic and antimicrobial agents. A detailed kinetic and spectroscopic study has been performed to probe the binding of a triazole-based inhibitor and a bestatin-based inhibitor to both Mn(II)- and Co(II)-loaded type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs. Both inhibitors were found to be moderate competitive inhibitors. The triazole-type inhibitor was found to interact with both active-site metal ions, while the bestatin-type inhibitor was capable of switching its mode of binding depending on the metal in the active site and the type of MetAP enzyme.     

Thermodynamic basis for the stabilities of three CutA1s from Pyrococcus horikoshii,Thermus thermophilus, and Oryza sativa, with unusually high denaturation temperatures

In order to elucidate the stabilization mechanism of CutA1 from Pyrococcus horikoshii (PhCutA1) with a denaturation temperature of nearly 150 degrees C, GuHCl denaturation and heat denaturation were examined at neutral and acidic pHs. As a comparison, CutA1 proteins from Thermus thermophilus (TtCutA1) and Oryza sativa (OsCutA1) were also examined, which have lower optimum growth temperatures of 75 and 28 degrees C, respectively, than that (98 degrees C) of P. horikoshii. GuHCl-induced unfolding and refolding curves of the three proteins showed hysteresis effects due to an unusually slow unfolding rate. The midpoints of refolding for PhCutA1, TtCutA1 and OsCutA1 were 5.7 M, 3.3 M, and 2.3 M GuHCl, respectively, at pH 8.0 and 37 degrees C. DSC experiments with TtCutA1 and OsCutA1 showed that the denaturation temperatures were remarkably high, 112.8 and 97.3 degrees C, respectively, at pH 7.0 and that the good heat reversibility was amenable to thermodynamic analyses. At acidic pH, TtCutA1 showed higher stability to both heat and denaturant than PhCutA1. Combined with the data for DSC and denaturant denaturation, the unfolding Gibbs energy of PhCutA1 could be depicted as a function of temperature. It was experimentally revealed that (1) the unusually high stability of PhCutA1 basically originates from a common trimer structure of the three proteins, (2) the stability of PhCutA1 is superior to those of the other two CutA1s over all temperatures above 0 degrees C at neutral pH, due to the decrease in both enthalpy and entropy, and (3) ion pairs of PhCutA1 contribute to the unusually high stability at neutral pH.     

Non-cognate enzyme-DNA complex: structural and kinetic analysis of EcoRV endonuclease bound to the EcoRI recognition site GAATTC

The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity.     

Crystal complex structures reveal how substrate is bound in the -4 to the +2 binding sites of Humicola grisea Cel12A

As part of an ongoing enzyme discovery program to investigate the properties and catalytic mechanism of glycoside hydrolase family 12 (GH 12) endoglucanases, a GH family that contains several cellulases that are of interest in industrial applications, we have solved four new crystal structures of wild-type Humicola grisea Cel12A in complexes formed by soaking with cellobiose, cellotetraose, cellopentaose, and a thio-linked cellotetraose derivative (G2SG2). These complex structures allow mapping of the non-covalent interactions between the enzyme and the glucosyl chain bound in subsites -4 to +2 of the enzyme, and shed light on the mechanism and function of GH 12 cellulases. The unhydrolysed cellopentaose and the G2SG2 cello-oligomers span the active site of the catalytically active H.grisea Cel12A enzyme, with the pyranoside bound in subsite -1 displaying a S31 skew boat conformation. After soaking in cellotetraose, the cello-oligomer that is found bound in site -4 to -1 contains a beta-1,3-linkage between the two cellobiose units in the oligomer, which is believed to have been formed by a transglycosylation reaction that has occurred during the ligand soak of the protein crystals. The close fit of this ligand and the binding sites occupied suggest a novel mixed beta-glucanase activity for this enzyme.     

Control of the nitrile-hydrolyzing enzyme activity in Rhodococcus rhodochrous IFO 15564: preferential action of nitrile hydratase and amidase depending on the reaction condition factors and its application to the one-pot preparation of amides from aldehydes

The reaction conditions towards the preferential action of either nitrile hydratase or amidase in the harvested whole cells of Rhodococcus rhodochrous IFO 15564 were elaborated. The amidase showed higher heat tolerance than the nitrile hydratase and, at 45 °C the amidase worked exclusively. DMSO assisted the preferential action of nitrile hydratase, however, at more than 30% (v/v) addition of DMF, the nitrile hydratase activity was completely lost and only amidase worked. A one-pot chemo-enzymatic conversion of aldehydes to amides [(1) aq. NH₃, I₂, DMSO; (2) Na₂S₂O₃; (3) harvested cells of R. rhodochrous] was established. Under these reaction conditions, most of the amidase was lost, and the incubation of the firstly formed intermediates, nitriles in aq. NH₃ was responsible for the selective inhibition of amidase. The freezing of harvested cells in an exhaustively deionized environment provided a long-term preservable “ready to use” for the organic chemist.     

Nuclear magnetic resonance spectroscopy with the stringent substrate rhodanese bound to the single-ring variant SR1 of the E. coli chaperonin GroEL

Nuclear magnetic resonance (NMR) observation of the uniformly (2) H,(15) N-labeled stringent 33-kDa substrate protein rhodanese in a productive complex with the uniformly (14) N-labeled 400 kDa single-ring version of the E. coli chaperonin GroEL, SR1, was achieved with the use of transverse relaxation-optimized spectroscopy, cross-correlated relaxation-induced polarization transfer, and cross-correlated relaxation-enhanced polarization transfer. To characterize the NMR-observable parts of the bound rhodanese, coherence buildup rates by different magnetization transfer mechanisms were measured, and effects of covalent crosslinking of the rhodanese to the apical binding surface of SR1 were investigated. The results indicate that the NMR-observable parts of the SR1-bound rhodanese are involved in intracomplex rate processes, which are not related to binding and release of the substrate protein from the SR1 binding surface. Rather, they correspond to mobility of the stably bound substrate, which thus appears to include flexibly disordered polypeptide segments devoid of long-lived secondary structures or tertiary folds, as was previously observed also with the smaller substrate human dihydrofolate reductase.     

Systematic evaluation of amide bioisosteres leading to the discovery of novel and potent thiazolylimidazolidinone inhibitors of SCD1 for the treatment of metabolic diseases

Several five- and six-membered heterocycles were introduced to replace the C2-position amide bond of the original 2-aminothiazole-based hit compound 5. Specifically, replacement of the amide bond with an imidazolidinone moiety yielded a novel and potent thiazolylimidazolidinone series of SCD1 inhibitors. XEN723 (compound 22) was identified after optimization of the thiazolylimidazolidinone series. This compound demonstrated a 560-fold improvement in in vitro potency and reduced plasma desaturation indices in a dose dependent manner, with an EC₅₀ of 4.5 mg/kg.     

Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4

A semi-purified nitrile hydratase from Rhodococcus erythropolis A4 was applied to biotransformations of 3-oxonitriles 1a–4a, 3-hydroxy-2-methylenenitriles 5a–7a, 4-hydroxy-2-methylenenitriles 8a–9a, 3-hydroxynitriles 10a–12a and 3-acyloxynitrile 13a into amides 1b–13b. Cross-linked enzyme aggregates (CLEAs) with nitrile hydratase and amidase activities (88% and 77% of the initial activities, respectively) were prepared from cell-free extract of this microorganism and used for nitrile hydration in presence of ammonium sulfate, which selectively inhibited amidase activity. The genes nha1 and nha2 coding for and β subunits of nitrile hydratase were cloned and sequenced.     

Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation

The enzymatic degradation of the plant cell wall is central both to the natural carbon cycle and, increasingly, to environmentally friendly routes to biomass conversion, including the production of biofuels. The plant cell wall is a complex composite of cellulose microfibrils embedded in diverse polysaccharides collectively termed hemicelluloses. Xyloglucan is one such polysaccharide whose hydrolysis is catalyzed by diverse xyloglucanases. Here we present the structure of the Clostridium thermocellum xyloglucanase Xgh74A in both apo and ligand-complexed forms. The structures, in combination with mutagenesis data on the catalytic residues and the kinetics and specificity of xyloglucan hydrolysis reveal a complex subsite specificity accommodating seventeen monosaccharide moieties of the multibranched substrate in an open substrate binding terrain.     

Interaction between bound water molecules and local protein structures: A statistical analysis of the hydrogen bond structures around bound water molecules

Water molecules play an important role in protein folding and protein interactions through their structural association with proteins. Examples of such structural association can be found in protein crystal structures, and can often explain protein functionality in the context of structure. We herein report the systematic analysis of the local structures of proteins interacting with water molecules, and the characterization of their geometric features. We first examined the interaction of water molecules with a large local interaction environment by comparing the preference of water molecules in three regions, namely, the protein–protein interaction (PPI) interfaces, the crystal contact (CC) interfaces, and the non‐interfacial regions. High preference of water molecules to the PPI and CC interfaces was found. In addition, the bound water on the PPI interface was more favorably associated with the complex interaction structure, implying that such water‐mediated structures may participate in the shaping of the PPI interface. The pairwise water‐mediated interaction was then investigated, and the water‐mediated residue–residue interaction potential was derived. Subsequently, the types of polar atoms surrounding the water molecules were analyzed, and the preference of the hydrogen bond acceptor was observed. Furthermore, the geometries of the structures interacting with water were analyzed, and it was found that the major structure on the protein surface exhibited planar geometry rather than tetrahedral geometry. Several previously undiscovered characteristics of water–protein interactions were unfolded in this study, and are expected to lead to a better understanding of protein structure and function. Proteins 2016; 84:43–51. © 2015 Wiley Periodicals, Inc.