The alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a novel cobalamin-dependent mutase pathway

Abstract

Fuel oxygenates such as methyl and ethyl tert-butyl ether (MTBE and ETBE, respectively) are degraded only by a limited number of bacterial strains. The aerobic pathway is generally thought to run via tert-butyl alcohol (TBA) and 2-hydroxyisobutyrate (2-HIBA), whereas further steps are unclear. We have now demonstrated for the newly isolated beta-proteobacterial strains L108 and L10, as well as for the closely related strain CIP I-2052, that 2-HIBA was degraded by a cobalamin-dependent enzymatic step. In these strains, growth on substrates containing the tert-butyl moiety, such as MTBE, TBA, and 2-HIBA, was strictly dependent on cobalt, which could be replaced by cobalamin. Tandem mass spectrometry identified a 2-HIBA-induced protein with high similarity to a peptide whose gene sequence was found in the finished genome of the MTBE-degrading strain Methylibium petroleiphilum PM1. Alignment analysis identified it as the small subunit of isobutyryl-coenzyme A (CoA) mutase (ICM; EC 5.4.99.13), which is a cobalamin-containing carbon skeleton-rearranging enzyme, originally described only in Streptomyces spp. Sequencing of the genes of both ICM subunits from strain L108 revealed nearly 100% identity with the corresponding peptide sequences from M. petroleiphilum PM1, suggesting a horizontal gene transfer event to have occurred between these strains. Enzyme activity was demonstrated in crude extracts of induced cells of strains L108 and L10, transforming 2-HIBA into 3-hydroxybutyrate in the presence of CoA and ATP. The physiological and evolutionary aspects of this novel pathway involved in MTBE and ETBE metabolism are discussed.

FIG. 1.

Proposed pathways for the aerobic degradation of the fuel oxygenates MTBE and ETBE (16, 44, 46).

FIG. 2.

Growth on 2-HIBA in mineral salt medium supplemented with either 100 μg of cyanocobalamin or 50 μg of cobalt ions per liter (filled and open symbols, respectively). 2-HIBA concentrations (circles) and dry weight biomass (triangles) were measured for batch cultures of strain L10 using a cobalt- and cobalamin-deficient inoculum. The data represent the mean values and SD of four replicates.

FIG. 3.

Degradation of TBA (a) and concomitant accumulation of 2-HIBA (b) in mineral salt medium supplemented with 1, 5, or 50 μg of cyanocobalamin per liter and using a cobalt- and cobalamin-deficient culture of strain L10. The data represent the mean values and SD of four replicates.

FIG. 4.

Transformation of 2-HIBA into 3-hydroxybutyrate in cell extracts of 2-HIBA-grown cells of strain L108. The complete assay contained 2-HIBA, CoA, and ATP, whereas in the other cases either CoA or ATP was omitted. The SD of the replicates was within 5% (not shown).

FIG. 5.

A CLUSTAL W alignment of a 30-amino-acid segment of the ICM large subunit (IcmA) of Methylibium petroleiphilum PM1 (ZP_00242470), with the corresponding sequences of the four closest BLAST matches using the complete IcmA sequence of strain PM1 as a query against the NCBI database (in descending order): Rhodobacter sphaeroides ATCC 17029 (EAP67072), Xanthobacter autotrophicus Py2 (EAS17594), Nocardioides sp. strain JS614 (EAO08692), and Roseovarius sp. strain 217 (EAQ26421) (for complete search results, see the supplemental material). For comparison, alignment with the corresponding ICM and MCM sequences of S. cinnamonensis (AJ246005) and P. shermanii (X14965), respectively, is also shown. Amino acids that are conserved in all sequences are indicated under the sequence by asterisk. Residues in boldface represent the reactive site position proposed to play an important role in substrate binding and reaction mechanism of both ICM and MCM (32, 39, 50).

FIG. 6.

Possible pathways for 2-HIBA degradation using a cobalamin-dependent carbon skeleton-rearranging step catalyzed by ICM. Initially, 2-HIBA may be activated to 2-hydroxyisobutyryl-CoA by acyl-CoA synthetases or CoA transferases.