Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.Many eubacteria are able to grow on linear alkanes by virtue of alkane hydroxylases (AHs) that activate alkanes to 1-alkanols. These are then further metabolized by alcohol and aldehyde dehydrogenases to fatty acids, which enter the central metabolism (34). AHs belong to several different oxygenase classes. Shortchain-length (C 2 to C 4 )-alkane degraders possess enzymes related to the soluble and particulate methane monooxygenases (34), while integral membrane nonheme iron AHs related to AlkB of Pseudomonas putida GPo1 were found in medium-chain-length (MCL) (C 5 to C 11 )-and especially in long-chain-length (LCL) (ՆC 12 )-alkane-degrading Alpha-, Beta-, and Gammaproteobacteria and high-GϩC gram-positive bacteria (34, 37).Several strains in our collection of alkane degraders grow well on MCL alkanes but could not be shown to contain AlkB homologs that act on MCL alkanes (some of these strains do contain AlkB homologs that hydroxylate LCL alkanes); these strains include the following. (i) Gordonia sp. strain 7E1C (Rhodococcus rhodochrous NCIMB 12566) is of interest because it is able to oxidize substituted phenoxy propane to phenoxy propanoic acids when pregrown on n-alkanes (17). It is known to contain an alkane-inducible cytochrome P450 (2).(ii) For Rhodococcus erythropolis NRRL B-16531 and Q15, none of the integral membrane AHs cloned from these isolates could be shown to act on MCL alkanes (39), even though these and other R. erythropolis isolates grow well on such alkanes (38). (iii) Of several hexane-degrading strains isolated from a trickling-bed bioreactor (32, 39), only 2 of 15 strains tested contained AlkB homologs that oxidize MCL alkanes (J. B. van Beilen et al., unpublished data). This left 13 strains for which growth on hexane initially could not be attributed to known enzyme systems. One of these strains, Sphingomonas sp. strain HXN-200, contains a soluble alkane hydroxylase, which is proposed to be responsible for a range of useful hydroxylation and epoxidation reactions of cyclic compounds such as pyrrolidines, pyrrolidinones, azetidines, azetidinones, piperidines, and piperidinones (4-6, 24, 25). Mycobacterium sp. strain HXN-1500, a strain able to convert limonene to perillyl alcohol (35), was found to contain a soluble alkane hydroxylase that is closely related to the Acinetobacter sp. strain EB104 hexane hydroxylase (CYP...

CCA-adding enzymes are specialized polymerases that add a specific sequence (C-C-A) to tRNA 3 ends without requiring a nucleic acid template. In some organisms, CCA synthesis is accomplished by the collaboration of evolutionary closely related enzymes with partial activities (CC and A addition). These enzymes carry all known motifs of the catalytic core found in CCA-adding enzymes. Therefore, it is a mystery why these polymerases are restricted in their activity and do not synthesize a complete CCA terminus. Here, a region located outside of the conserved motifs was identified that is missing in CC-adding enzymes. When recombinantly introduced from a CCA-adding enzyme, the region restores full CCAadding activity in the resulting chimera. Correspondingly, deleting the region in a CCA-adding enzyme abolishes the A-incorporating activity, also leading to CC addition. The presence of the deletion was used to predict the CC-adding activity of putative bacterial tRNA nucleotidyltransferases. Indeed, two such enzymes were experimentally identified as CC-adding enzymes, indicating that the existence of the deletion is a hallmark for this activity. Furthermore, phylogenetic analysis of identified and putative CCadding enzymes indicates that this type of tRNA nucleotidyltransferases emerged several times during evolution. Obviously, these enzymes descend from CCA-adding enzymes, where the occurrence of the deletion led to the restricted activity of CC addition. A-adding enzymes, however, seem to represent a monophyletic group that might also be ancestral to CCA-adding enzymes. Yet, experimental data indicate that it is possible that A-adding activities also evolved from CCA-adding enzymes by the occurrence of individual point mutations.tRNA maturation ͉ CCA-adding enzyme ͉ flexible loop

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)-associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non-tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H2O2). A population of HCEC survived H2O2-induced oxidative stress via JNK-dependent cell cycle arrests. Caspases, p21WAF1 and γ-H2AX were identified as JNK-regulated proteins. Up-regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan-caspase inhibitor Z-VAD-FMK caused up-regulation of γ-H2AX, a DNA-damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G1-phase as first response to oxidative stress and increased S-phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non-apoptotic function by promoting cells through G1- and S-phase by overriding the G1/S- and intra-S checkpoints despite DNA-damage. This led to the accumulation of cells in the G2/M-phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ-H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase-dependent proteolytic degradation of the DNA-damage checkpoint protein ATM that is upstream of γ-H2AX. As a consequence, undetected DNA-damage and increased proliferation were found in repeatedly H2O2-exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non-apoptotic function of caspases. Overexpression of upstream p-JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.

Showing a high sequence similarity, the evolutionary closely related bacterial poly(A) polymerases (PAP) and CCA-adding enzymes catalyze quite different reactions—PAP adds poly(A) tails to RNA 3′-ends, while CCA-adding enzymes synthesize the sequence CCA at the 3′-terminus of tRNAs. Here, two highly conserved structural elements of the corresponding Escherichia coli enzymes were characterized. The first element is a set of amino acids that was identified in CCA-adding enzymes as a template region determining the enzymes' specificity for CTP and ATP. The same element is also present in PAP, where it confers ATP specificity. The second investigated region corresponds to a flexible loop in CCA-adding enzymes and is involved in the incorporation of the terminal A-residue. Although, PAP seems to carry a similar flexible region, the functional relevance of this element in PAP is not known. The presented results show that the template region has an essential function in both enzymes, while the second element is surprisingly dispensable in PAP. The data support the idea that the bacterial PAP descends from CCA-adding enzymes and still carries some of the structural elements required for CCA-addition as an evolutionary relic and is now fixed in a conformation specific for A-addition.

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