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Journal of Bacteriology, January 2003, p. 41-50, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.41-50.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of the 4-Carboxy-4-Hydroxy-2-Oxoadipate Aldolase Gene and Operon Structure of the Protocatechuate 4,5-Cleavage Pathway Genes in Sphingomonas paucimobilis SYK-6

Hirofumi Hara,1 Eiji Masai,1* Keisuke Miyauchi,1 Yoshihiro Katayama,2 and Masao Fukuda1

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188,1 and Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan2

Received 29 July 2002/ Accepted 4 October 2002

The protocatechuate (PCA) 4,5-cleavage pathway is the essential metabolic route for degradation of low-molecular-weight products derived from lignin by Sphingomonas paucimobilis SYK-6. In the 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), 4-oxalomesaconate hydratase (ligJ), and a part of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligK gene, which encodes 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase. The ligK gene was located 1,183 bp upstream of ligI and transcribed in the same direction as ligI. We also found the ligR gene encoding a LysR-type transcriptional activator, which was located 174 bp upstream of ligK. The ligK gene consists of a 684-bp open reading frame encoding a polypeptide with a molecular mass of 24,131 Da. The deduced amino acid sequence of ligK showed 57 to 88% identity with those of the corresponding genes recently reported in Sphingomonas sp. strain LB126, Comamonas testosteroni BR6020, Arthrobacter keyseri 12B, and Pseudomonas ochraceae NGJ1. The ligK gene was expressed in Escherichia coli, and the gene product (LigK) was purified to near homogeneity. Electrospray-ionization mass spectrometry indicated that LigK catalyzes not only the conversion of CHA to pyruvate and oxaloacetate but also that of oxaloacetate to pyruvate and CO2. LigK is a hexamer, and its isoelectric point is 5.1. The Km for CHA and oxaloacetate are 11.2 and 136 µM, respectively. Inactivation of ligK in S. paucimobilis SYK-6 resulted in the growth deficiency of vanillate and syringate, indicating that ligK encodes the essential CHA aldolase for catabolism of these compounds. Reverse transcription-PCR analysis revealed that the PCA 4,5-cleavage pathway genes of S. paucimobilis SYK-6 consisted of four transcriptional units, including the ligK-orf1-ligI-lsdA cluster, the ligJAB cluster, and the monocistronic ligR and ligC genes.

* Corresponding author. Mailing address: Department of Bioengineering, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata 940-2188, Japan. Phone: 81-258-47-9428. Fax: 81-258-47-9450. E-mail: emasai{at}vos.nagaokaut.ac.jp.

Journal of Bacteriology, January 2003, p. 41-50, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.41-50.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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