Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.2.2.10 (PNL)
 341 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The three-dimensional structure of Aspergillus niger pectin lyase B (PLB) has been determined by crystallographic techniques at a resolution of 1.7 A. The model, with all 359 amino acids and 339 water molecules, refines to a final crystallographic R factor of 16.5%. The polypeptide backbone folds into a large right-handed cylinder, termed a parallel beta helix. Loops of various sizes and conformations protrude from the central helix and probably confer function. The largest loop of 53 residues folds into a small domain consisting of three antiparallel beta strands, one turn of an alpha helix, and one turn of a 3(10) helix. By comparison with the structure of Erwinia chrysanthemi pectate lyase C (PelC), the primary sequence alignment between the pectate and pectin lyase subfamilies has been corrected and the active site region for the pectin lyases deduced. The substrate-binding site in PLB is considerably less hydrophilic than the comparable PelC region and consists of an extensive network of highly conserved Trp and His residues. The PLB structure provides an atomic explanation for the lack of a catalytic requirement for Ca2+ in the pectin lyase family, in contrast to that found in the pectate lyase enzymes. Surprisingly, however, the PLB site analogous to the Ca2+ site in PelC is filled with a positive charge provided by a conserved Arg in the pectin lyases. The significance of the finding with regard to the enzymatic mechanism is discussed.
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PMID:The tree-dimensional structure of aspergillus niger pectin lyase B at 1.7-A resolution. 944 37

Most structures of neutral lipases and esterases have been found to adopt the common alpha/beta hydrolase fold and contain a catalytic Ser-His-Asp triad. Some variation occurs in both the overall protein fold and in the location of the catalytic triad, and in some enzymes the role of the aspartate residue is replaced by a main-chain carbonyl oxygen atom. Here, we report the crystal structure of pectin methylesterase that has neither the common alpha/beta hydrolase fold nor the common catalytic triad. The structure of the Erwinia chrysanthemi enzyme was solved by multiple isomorphous replacement and refined at 2.4 A to a conventional crystallographic R-factor of 17.9 % (R(free) 21.1 %). This is the first structure of a pectin methylesterase and reveals the enzyme to comprise a right-handed parallel beta-helix as seen in the pectinolytic enzymes pectate lyase, pectin lyase, polygalacturonase and rhamnogalacturonase, and unlike the alpha/beta hydrolase fold of rhamnogalacturonan acetylesterase with which it shares esterase activity. Pectin methylesterase has no significant sequence similarity with any protein of known structure. Sequence conservation among the pectin methylesterases has been mapped onto the structure and reveals that the active site comprises two aspartate residues and an arginine residue. These proposed catalytic residues, located on the solvent-accessible surface of the parallel beta-helix and in a cleft formed by external loops, are at a location similar to that of the active site and substrate-binding cleft of pectate lyase. The structure of pectin methylesterase is an example of a new family of esterases.
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PMID:Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site. 1116 5

A pectin lyase gene (pnl) of Pseudomonas marginalis was cloned and overexpressed in Escherichia coli BL21(DE3). The pnl gene was amplified by PCR, inserted into pET29c with a six-His tag and the overproduced active enzyme was purified almost to homogeneity using a Ni(2+)-nitrilotriacetate-agarose column. The purified pectin lyase (PNL; EC 4.2.2.10, family 1) is inhibited by NAD+ (at concentrations above 0.25 mM), NADH or dithiothreitol. Evidence for the existence of a heat-labile protein inhibitor of PNL is also reported. The DNA-binding ability of PNL was demonstrated by DNA-retardation experiments. The partially purified enzyme was incubated with plasmid DNA and the complex was shifted to a higher molecular mass. Analysis of the electroeluted proteins from the protein-DNA complex revealed that one of the electroeluted protein bands was PNL. Antibodies against the overexpressed PNL were also prepared and partially purified.
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PMID:Overexpression of the pectin lyase gene of Pseudomonas marginalis in Escherichia coli and purification of the active enzyme. 1263 Sep 8

The human NUDT12 Nudix hydrolase has been expressed in insect cells from a baculovirus vector as a His-tagged recombinant protein. In vitro, it efficiently hydrolyses NAD(P)H to NMNH and AMP (2',5'-ADP), and diadenosine diphosphate to AMP. It also has activity towards NAD(P)(+), ADP-ribose and diadenosine triphosphate. K (m) values for NADH, NADPH and NAD(+) are 11, 16 and 190 microM and k (cat) values are 11, 16 and 10.5 s(-1) respectively. Thus, like other NADH diphosphatases of the Nudix family, NUDT12 has a marked substrate preference for the reduced nicotinamide nucleotides. Optimal activity was supported by 50 microM Mn(2+) ions in vitro, with 3-fold lower activity at 0.4 mM Mg(2+). Expression of NUDT12 as a C-terminal fusion to green fluorescent protein revealed that it was targeted to peroxisomes by the C-terminal tripeptide PNL acting as a novel type 1 peroxisomal targeting signal. Deletion of PNL resulted in diffuse cellular fluorescence. In addition, C-terminal, but not N-terminal, fusions with or without the PNL signal accumulated in large, unidentified cytoplasmic structures. NUDT12 may act to regulate the concentration of peroxisomal nicotinamide nucleotide cofactors required for oxidative metabolism in this organelle.
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PMID:Mammalian NADH diphosphatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein. 1279 Jul 96