Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2·0Åresolution

Romão, M.J.; Turk, Dušan; Gomis‐Rüth, F. Xavier; Huber, Robert; Schumacher, Günter; Möllering, Hans; L, Rüssmann

doi:10.1016/0022-2836(92)91056-u

Cited by 40 publications

(55 citation statements)

References 28 publications

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“…Presumably, the initial products are carbamate and aminoacrylate, which are known to hydrolyze spontaneously. RutB is homologous to the ureidopropionase enzyme of the reductive pathway for pyrimidine ring degradation (39,54), and its closest homologue is carbamoylsarcosine amidohydrolase (25,32): both release CO 2 and NH 3 via carbamate. For reasons given below, we propose that RutB be called peroxyureidoacrylate/ureidoacrylate amido hydrolase.…”

Section: Discussionmentioning

confidence: 99%

The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

Kim

Pelton

Inwood³

et al. 2010

J Bacteriol

103

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The Rut pathway is composed of seven proteins, all of which are required by Escherichia coli K-12 to grow on uracil as the sole nitrogen source. The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. RutA works in conjunction with a flavin reductase (RutF or a substitute) to catalyze a novel reaction. It directly cleaves the uracil ring between N-3 and C-4 to yield ureidoacrylate, as established by both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Although ureidoacrylate appears to arise by hydrolysis, the requirements for the reaction and the incorporation of 18 O at C-4 from molecular oxygen indicate otherwise. Mass spectrometry revealed the presence of a small amount of product with the mass of ureidoacrylate peracid in reaction mixtures, and we infer that this is the direct product of RutA. In vitro RutB cleaves ureidoacrylate hydrolytically to release 2 mol of ammonium, malonic semialdehyde, and carbon dioxide. Presumably the direct products are aminoacrylate and carbamate, both of which hydrolyze spontaneously. Together with bioinformatic predictions and published crystal structures, genetic and physiological studies allow us to predict functions for RutC, -D, and -E. In vivo we postulate that RutB hydrolyzes the peracid of ureidoacrylate to yield the peracid of aminoacrylate. We speculate that RutC reduces aminoacrylate peracid to aminoacrylate and RutD increases the rate of spontaneous hydrolysis of aminoacrylate. The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. RutG appears to be a uracil transporter.The rut (pyrimidine utilization) operon of Escherichia coli K-12 contains seven genes (rutA to -G) (31,38). A divergently transcribed gene (rutR) codes for a regulator. The RutR regulator is now known to control not only pyrimidine degradation but also pyrimidine biosynthesis and perhaps a number of other things (44,45). In the presence of uracil, RutR repression of the rut operon is relieved.Superimposed on specific regulation of the rut operon by RutR is general control by nitrogen regulatory protein C (NtrC), indicating that the function of the Rut pathway is to release nitrogen (31, 59). The rut operon was discovered in E. coli K-12 as one of the most highly expressed operons under NtrC control. In vivo it yields 2 mol of utilizable nitrogen per mol of uracil or thymine and 1 mol of 3-hydroxypropionic acid or 2-methyl 3-hydroxypropionic acid, respectively, as a waste product (Fig. 1). Waste products are excreted into the medium. (Lactic acid is 2-hydroxypropionic acid.) Wild-type E. coli K-12 can use uridine as the sole nitrogen source at temperatures up to 22°C but not higher. It is chemotactic to pyrimidine bases by means of the methyl-accepting chemoreceptor TAP (taxis toward dipeptides), but this response is not temperature dependent (30).In the known reductive and oxidative pathways for degradation of the pyrimidine ring (22, 48, 52), the C-5-C-6 double bond ...

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Section: Discussionmentioning

confidence: 99%

The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

Kim

Pelton

Inwood³

et al. 2010

J Bacteriol

103

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“…Analysis of all the data presented above has led us to a hypothesis that the structural motifs having unique overall folds can fold independently of the 187 ; CSH, N-carbamoylsarcosine amidohydrolase 188 ; phosphorylase a, glycogen phosphorylase a 189 . Figure 4.…”

Section: Discussionmentioning

confidence: 98%

Structural trees for protein superfamilies

Ефимов

1997

Proteins

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Structural trees for large protein superfamilies, such as beta proteins with the aligned beta sheet packing, beta proteins with the orthogonal packing of alpha helices, two-layer and three-layer alpha/beta proteins, have been constructed. The structural motifs having unique overall folds and a unique handedness are taken as root structures of the trees. The larger protein structures of each superfamily are obtained by a stepwise addition of alpha helices and/or beta strands to the corresponding root motif, taking into account a restricted set of rules inferred from known principles of the protein structure. Among these rules, prohibition of crossing connections, attention to handedness and compactness, and a requirement for alpha helices to be packed in alpha-helical layers and beta strands in beta layers are the most important. Proteins and domains whose structures can be obtained by stepwise addition of alpha helices and/or beta strands to the same root motif can be grouped into one structural class or a superfamily. Proteins and domains found within branches of a structural tree can be grouped into subclasses or subfamilies. Levels of structural similarity between different proteins can easily be observed by visual inspection. Within one branch, protein structures having a higher position in the tree include the structures located lower. Proteins and domains of different branches have the structure located in the branching point as the common fold.

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“…The product of pehA is related to a hydrolase (CSHase) from Arthrobacter sp. which catalyzes the hydrolysis of N-carbamoylsarcosine to sarcosine, CO 2 , and NH 3 (77). The cysteine at residue 159 proposed to be the catalytic nucleophile in CSHase (as residue 177) is conserved.…”

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confidence: 99%

Plasmid-Encoded Phthalate Catabolic Pathway in Arthrobacter keyseri 12B

2001

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Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown Arthrobacter keyseri (formerly Micrococcus sp.) 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates). Because these products lack a carboxyl group at the 2 position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated. When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates. This chromogenic response was subsequently used to identify recombinant Escherichia coli strains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130-kbp plasmid pRE1 of strain 12B. Beginning with the initially cloned 8.14-kbp PstI fragment of pRE824 as a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases. From these plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units: tnpR, pcm operon, ptr genes, pehA, norA fragment, and pht operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively. Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant E. coli strains.

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Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2·0Åresolution

Cited by 40 publications

References 28 publications

The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

Structural trees for protein superfamilies

Plasmid-Encoded Phthalate Catabolic Pathway in Arthrobacter keyseri 12B

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