Supplementary materials
Identification and characterization of 2-keto-3-deoxy-L-rhamnonate
dehydrogenase belonging to the MDR superfamily from the
thermoacidophilic bacterium Sulfobacillus thermosulfidooxidans:
implications to L-rhamnose metabolism in archaea
Jungdon Bae1, Suk Min Kim1, Sun Bok Lee1,2*
Department of Chemical Engineering1, Graduate School of Engineering Mastership2, Pohang
University of Science and Technology, San 31, Hyoja Dong, Pohang 790-784, Korea
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Table S1. Characteristics of four Rha_NMP genes in Sulfobacillus thermosulfidooxidans AT-1 and the
primers used for cloning
Locus tag
Sulth_3557
Sulth_3558
Sulth_3559
Sulth_3560
COG category
COG1063
COG0179
COG4948
Annotation
L-threonine 3dehydrogenase
ureidoglycolate
lyase
COG1028
3-oxoacyl-[acylcarrier-protein]
reductase
Predicted protein
function
2-keto-3-deoxy-Lrhamnonate
dehydrogenase
2,4-diketo-3-deoxyL-rhamnonate
hydrolase
L-rhamnose
dehydrogenase
L-rhamnonate
dehydratase
Superfamily
MDR
fumarylacetoacetate
hydrolase
SDR
ORF (bp)
AA (ea)
MW (Da)
GC (%)
996
331
35,694
49.2
900
299
33,362
45.1
762
253
27,114
47.4
mandelate
racemase/muconate
lactonizing enzyme
1,194
397
44,500
45.4
GAGGATCCATGAA
AACTTTAACATGG
ACGGC
CCAAGCTTCTAAA
ATGTTAAGATGATT
TTG
BamHI/HindIII
GAGGATCCATGAA
ACTAGCCAGTGCC
ATAG
CCAAGCTTTCATG
ACTTTGTGTTCGC
T
BamHI/HindIII
GTTGGATCCATGG
CATTAACAGGAAA
AGTGG
TATAAGCTTTCACT
GTAAATTCACAAA
CAATC
BamHI/HindIII
TTAGGATCCATGA
AAAATAACCTGAA
AATTGTTC
CCCTGCAGCTAAG
ACGGCGCAGAGG
AAACG
BamHI/PstI
Primer (forward)
Primer (reverse)
Restriction sites
Expression vector
PCR
L-rhamnonate
dehydratase
pQE-80L
33 cycles, denaturation at 94°C for 30 sec, annealing at 55°C for 40 sec, and
polymerization at 72°C for 40 sec.
-2-
Figure S1. SDS-PAGE of recombinant proteins purified from the E. coli DH5α transformants. A.
Sulth_3559 (St_RhaDH). B. Sulth_3557 (St_KDRDH). Lane M, Tech & Innovation ACCU prestained
protein marker; lane 1, supernatant protein sample after sonication; lane 2, precipitated protein sample
after sonication lane 3, protein sample purified by Ni-NTA affinity chromatography; lane 4, protein
sample purified by Q-Sepharose FF chromatography. The amount of protein loaded onto a gel was 15
µg. The arrows indicate Sulth_3559 (A) and Sulth_3557 (B), respectively.
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Figure S2. Kinetic parameter determination of the recombinant St_KDRDH. (A) Five different LKDR concentrations (0.1-5 mM) in the presence of 1 mM NAD+, (B) six different NAD+
concentrations in the presence of 1 mM L-KDR, (C) six different BDO concentrations (0.1-100 mM)
in the presence of 1 mM NAD+, and (D) six different NAD+ concentrations in the presence of 50 mM
BDO. Apparent Vmax and Km values were determined by the Lineweaver–Burk plot using SigmaPlot
(version 10.0). Michaelis–Menten plots are shown in the inset.
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