Identification of a hydratase and a class II aldolase involved in biodegradation of the organic solvent tetralin

Abstract

Two new genes whose products are involved in biodegradation of the organic solvent tetralin were identified. These genes, designated thnE and thnF, are located downstream of the previously identified thnD gene and code for a hydratase and an aldolase, respectively. A sequence comparison of enzymes similar to ThnE showed the significant similarity of hydratases involved in biodegradation pathways to 4-oxalocrotonate decarboxylases and established four separate groups of related enzymes. Consistent with the sequence information, characterization of the reaction catalyzed by ThnE showed that it hydrated a 10-carbon dicarboxylic acid. The only reaction product detected was the enol tautomer, 2,4-dihydroxydec-2-ene-1,10-dioic acid. The aldolase ThnF showed significant similarity to aldolases involved in different catabolic pathways whose substrates are dihydroxylated dicarboxylic acids and which yield pyruvate and a semialdehyde. The reaction products of the aldol cleavage reaction catalyzed by ThnF were identified as pyruvate and the seven-carbon acid pimelic semialdehyde. ThnF and similar aldolases showed conservation of the active site residues identified by the crystal structure of 2-dehydro-3-deoxy-galactarate aldolase, a class II aldolase with a novel reaction mechanism, suggesting that these similar enzymes are class II aldolases. In contrast, ThnF did not show similarity to 4-hydroxy-2-oxovalerate aldolases of other biodegradation pathways, which are significantly larger and apparently are class I aldolases.

FIG. 1.

(A) Genomic region of strain TFA involved in tetralin biodegradation, showing the divergent thnC and thnD genes and the newly identified thnE and thnF genes. The arrows represent divergent operons. The triangles represent locations of KIXX insertions in mutants unable to grow on tetralin as the only carbon source. aa, amino acids. (B) Diagram of the thn genes cloned in the vector pBluescript II KS, which are transcribed from the φ10 promoter of phage T7 (large arrow).

FIG. 2.

Dendrogram showing the best tree obtained by the neighbor-joining method from the alignment of 27 sequences showing significant similarity to the hydratase ThnE. Scale bar = 0.1% divergence. The ThnE sequence is enclosed in a box. The GenBank accession numbers for other hydratases are as follows: EtbE/BphE Rsp RHA1, D78322; BphE Nar pNL1, XylI Nar pNL1, and XylJ Nar pNL1, AF079317; HpaH Sdu 2229, AF144422; HpaH Eco W, Z37980; HpcG Eco C, X81446; TbhG Bce AA1, AF001356; AtdE Asp YAA and AtdH Asp YAA, AB008831; DmpE Ppu CF600 and DmpH Ppu CF600, X60835; NahK Pst AN10 and NahL Pst AN10, AF039534; XylI Ppu PWW0, M94186; PhnK Psp DJ77, AF073359; CumE Pfl IP101, D63377; IpbE Ppu RE204, AF006691; BphE Psp LB400, X76500; BphX1 Pps KF707, D85853; XylJ Ppu PWW0, M64747; TodG Ppu F1, U09250; BphE Psp KKS102, D16407; PhnH Psp DJ77, U97697; Cmt Ppu F1, U24215; MhpD Eco K12, D86239; and MhpD Cte TA441, AB024335.

FIG. 3.

Characterized reactions of the tetralin catabolic pathway. 1,2-DHT, 1,2-dihydroxytetralin; HDDDA, 2-hydroxydec-2,4-diene-1,10-dioic acid; ODDA, 2-oxodec-4-ene-1,10-dioic acid.