Stereoselective catabolism of β-1 type lignin-derived aromaticisomers in Sphingobium sp. strain SYK-6
Eiji MASAI
Department of Materials Science and Bioengineering,
Nagaoka University of Technology
Research objectiveLignin, a plant cell wall component, is an aromatic polymer with complex linkages, andits abundance has made its use desirable. Currently, the metabolic capacity of bacteria is being
utilized to develop technology for the conversion of heterogeneous low-molecular weightaromatic compounds obtained through the chemical depolymerization of lignin into raw
materials for fabrication of functional polymers 1. A remarkable number of bacterial catabolicsystems essential for the development of this technology have been investigated. However, thecatabolic system for converting lignin-derived dimeric compounds with stereoisomers is notwell studied 2,3.
The β-1-type dimeric compound 1,2-diguaiacylpropane-1,3-diol (DGPD) exists as threo
and erythro stereoisomers, each of which contains enantiomers (Fig. 1). In Sphingobium sp.
strain SYK-6 cells, LigD and LigL, which are Cα-dehydrogenases involved in the degradationof β-O-4 dimeric compounds 4, play key roles in the oxidation of threo-DGPD (DGPD I andII) to the keto form (DGPD-keto I and II). Subsequently, DGPD-keto II is reduced to erythroDGPD (DGPD IV) in the presence of NADPH by LdpB, another Cα-dehydrogenase, andDGPD IV is rapidly degraded by an SYK-6 cell extract.

Fig. 1. The catabolic system for the conversion of the stereoisomers of β-1-type dimericcompounds in Sphingobium sp. SYK-6.
Thus, the DGPD stereoisomers are metabolized via a unique conversion system that
inverts the stereochemistry of the Cα hydroxyl group of threo-DGPD. In this study, we aimed
to determine the enzyme genes involved in the conversion of DGPD-keto I and erythro-DGPD

(DGPD III and IV) and their associated functions to gain insights into the catabolism of β-1type dimeric compounds.
Methods
The β-1 dimeric compounds were provided by Dr. Shojiro Hishiyama of the Forestry andForest Products Research Institute and Dr. Rui Katahira of the National Renewable Energy
Laboratory. Cosmid clones were isolated from a genomic library of SYK-6 constructed inSphingobium japonicum UT26S by selecting the transformants with erythro-DGPDconversion ability using HPLC analysis. Each gene was expressed using either the pET system
in E. coli or the pQF system in SYK-6. The gene products were purified using Ni-affinity
chromatography. The substrate conversion measurements and identification and quantificationof products were performed by HPLC, LC-MS, and chiral HPLC. The enzymatic activity ofLdpA was measured based on the increase in absorbance of the product DGPD-S at 333 nm.
The enzymatic activity of LdpC was measured based on the decrease in absorbance ofNADPH/NADH at 340 nm when DGPD-keto I and II were used as the substrates. Gene
disruption was performed by homologous recombination. Complementary plasmids for thegene-disruption strains were prepared by introducing each gene into pJB861.
Results
Identification and functional analysis of the erythro-DGPD-converting enzyme gene
A cosmid clone that conferred the erythro-DGPD conversion ability to host cells wasisolated from the genomic library of SYK-6. During this study, a group in the U.S. publisheda paper identifying an erythro-DGPD-converting enzyme gene lsdE from Novosphingobiumaromaticivorans DSM 12444 5. In the resulting cosmid, a gene showed an 80% amino acidsequence identity with DSM 12444 lsdE, which was designated as ldpA. LdpA produced in E.
coli was purified, and its enzymatic properties were investigated. LdpA catalyzed the Cγ
deformylation of erythro-DGPD (DGPD III and IV) accompanied by dehydroxylation at theCα-position, which led to the generation of a stilbene-type compound DGPD-S (Fig. 1). Incontrast, LdpA did not show threo-DGPD (DGPD I and II) conversion ability. An ldpAdisrupted strain of SYK-6 was defective in erythro-DGPD conversion, indicating that ldpA
was responsible for the conversion of erythro-DGPD. Additionally, DGPD-S was metabolized
via vanillate.
Identification of the DGPD-keto I-converting enzyme geneWe hypothesized that Cα-dehydrogenase of the short-chain dehydrogenase/reductase(SDR) family, which LdpB belongs to, is involved in DGPD-keto I reduction. Since DGPDketo I is converted in the presence of NADPH by the SYK-6 cell extract, six of the seven Cαdehydrogenase genes were expressed in E. coli, and the resulting cell extracts were incubatedwith DGPD-keto I in the presence of NADPH. HPLC analysis showed that none of therecombinant strains exhibited DGPD-keto I conversion activity. An unexpressed gene in E.
coli was expressed in SYK-6, and the gene product was purified using Ni-affinity
chromatography. However, no DGPD-keto I conversion activity was observed for this purified

enzyme.
ldpB is located upstream of ldpA in the SYK-6 genome. In addition to ldpB, two othergenes belonging to the SDR family are located around ldpA. Disruption strains for each gene
were generated, and the conversion ability of each mutant was determined by incubating therespective resting cells cultured in LB with DGPD-keto I. The amount of DGPD-keto I
converted by the ldpC-disrupted strain after 4 h of incubation was reduced to approximately
20% of that of the wild type. DGPD-keto I conversion ability was restored in the ldpCdisrupted strain complemented with ldpC. These results indicate that ldpC is primarily
responsible for the conversion of DGPD-keto I in SYK-6 cells.
Enzymatic function of LdpC
The cell extract of E. coli expressing ldpC was incubated with DGPD-keto I in thepresence of NADPH, and then the reaction mixture was analyzed using HPLC. The cell extract
converted DGPD-keto I into erythro-DGPD (Fig. 1). In contrast, the cell extract showed noconversion activity against DGPD-keto II. The specific activity of purified LdpC for DGPDketo I conversion in the presence of NADPH was 31 µmol/min/mg and that in the presence ofNADH was lower by less than 3%. These results indicate that LdpC exhibits NADPHdependent DGPD-keto I reduction activity.
Conclusion
The threo-DGPD isomers are oxidized by LigD and LigL in SYK-6 cells, followed byreduction via LdpC and LdpB, which invert the stereochemistry of the Cα hydroxyl group of
threo-DGPD. Since LdpA is specific to erythro-DGPD, the SYK-6 cells can catabolize allstereoisomers of the β-1 type dimer by converting the threo form to the erythro form.
References
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