Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of biuret to allophanate and ammonia. The kcat/KM of 1.7 × 105 M−1s−1 and the relatively low KM of 23 ± 4 μM together suggested that this enzyme acts uniquely on biuret physiologically. This is supported by the fact that of the 34 substrate analogs of biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for biuret. Biuret hydrolase does not react with carboxybiuret, the product of the enzyme immediately preceding biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway.
Vibrio furnissii M1 was recently reported to biosynthesize n-alkanes when grown on biopolymers, sugars, or organic acids (M. O. Park, J. Bacteriol. 187:1426-1429, 2005). In the present study, V. furnissii M1 was subjected to genomic analysis and studied biochemically. The sequence of the 16S rRNA gene and repetitive PCR showed that V. furnissii M1 was not identical to other V. furnissii strains tested, but the level of relatedness was consistent with its assignment as a V. furnissii strain. Pulsed-field gel electrophoresis showed chromosomal bands at approximately 3.2 and 1.8 Mb, similar to other Vibrio strains. Complete genomic DNA from V. furnissii M1 was sequenced with 21-fold coverage. Alkane biosynthetic and degradation genes could not be identified. Moreover, V. furnissii M1 did not produce demonstrable levels of n-alkanes in vivo or in vitro. In vivo experiments were conducted by growing V. furnissii M1 under different conditions, extracting with solvent, and analyzing extracts by gas chromatography-mass spectrometry. A highly sensitive assay was used for in vitro experiments with cell extracts and [14 C]hexadecanol. The data are consistent with the present strain being a V. furnissii with properties similar to those previously described but lacking the alkane-producing phenotype. V. furnissii ATCC 35016, also reported to biosynthesize alkanes, was found in the present study not to produce alkanes.The need for renewable energy sources will require the development of biofuel options other than ethanol. One excellent fuel option would be bio-alkanes. Alkanes comprise the major component of current petroleum-based fuels. A biological petroleum would be renewable and completely compatible with existing fuel infrastructure. Thus, considerable interest was generated by recent reports of high-level n-alkane formation by the bacterium Vibrio furnissii M1 (22, 23, 24).V. furnissii strains were recognized as a distinct species in 1983 (6). Other Vibrio species, such as V. cholerae (8) and V. parahaemolyticus (30), have been more extensively studied because of their significant pathogenicity in humans. Both of the latter species (12), along with V. vulnificus (7) and V. fischeri (25), have been subjected to genomic sequencing that has been completed and published. V. furnissii has been most extensively studied with respect to its physiological and genetic mechanisms of chitin degradation (3, 17). Marine vibrios are prominent chitinolytic organisms (18).Thus, the recent report of a V. furnissii strain biosynthesizing appreciable quantities of n-alkanes was unusual and interesting (22,23,24). The organism was isolated from activated sludge of a sewage disposal plant located in the Osaka prefecture of Japan (24). It was reported to produce a copious lipid layer that floated on top of liquid cultures. The culture lipids were found to consist of 48% alkanes (24), and the alkane-producing phenotype was the subject of three papers published between 2001 and 2005. The reports were significant for several reasons. First, n-a...
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