Characterization and gene cloning of l-xylulose reductase involved in l-arabinose catabolism from the pentose-fermenting fungus Rhizomucor pusillus

Figures & data

Fig. 1. l-Arabinose and d-xylose catabolism pathways in fungi and yeasts. LAR; l-arabinose reductase, LAD; l-arabinitol dehydrogenase, LXR; l-xylulose reductase, XR; d-xylose reductase, XDH; xylitol dehydrogenase.

Table 1. Purification steps of native RpLXR.

Step	Total protein (mg)	Total activity^a (U)	Specific activity^a (U/mg)	Yield (%)
Cell-free extracts	2700	368	0.137	100
(NH4)2SO4 precipitation	416	271	0.651	73.6
Butyl-Toyopearl	47.8	160	3.35	43.5
Gigapite	14.1	122	8.65	33.2
MonoQ HR 5/5	0.71	31.5	44.3	8.56
Superdex 200 HR 10/30	0.26	20.1	77.3	5.46

^a Xylitol and NADP⁺ were used as the substrate and coenzyme, respectively.

Fig. 2. SDS-PAGE of RpLXR during purification. Lanes. 1, Molecular-weight standards; 2, Cell-free extract; 3, Ammonium sulfate fractionation (50–90%); 4, Butyl-Toyopearl column chromatography; 5, Gigapite column chromatography; 6, MonoQ HR 5/5 column chromatography; 7, Superdex 200 HR 10/30 column chromatography.

Table 2. Substrate specificity of the native RpLXR.

Substrate	Relative activity (%)	Substrate	Relative activity (%)
Reduction		Oxidation
l-Xylulose	100	Xylitol	100
d-Xylulose	2.2	Glycerol	26.5
Dihydroxyacetone	165	d-Arabitol	6.9
Diacetyl	125	l-Arabitol	N.D.
d-Glyceraldehyde	1.5	Acetoin	6.9
dl-Glyceraldehyde	57.1	1,2-Propanediol	6.1
Hydroxyacetone	7.4	Ribitol	3.7
Acetone	N.D.	d-Mannitol	N.D.
Xylose	N.D.	d-Sorbitol	N.D.
d-Fructose	N.D.	Erythritol	N.D.

In the reductive reaction, the activity for ʟ-xylulose with NADPH, corresponding to 53.9 U/mg, was set to 100%.

In the oxidative reaction, the activity for xylitol with NADP⁺, corresponding to 56.2 U/mg, was set to 100%.

N.D.; not detected.

Table 3. Comparison of l-xylulose reductase from several fungi.

Strain	Enzyme	Mr. (native)	Mr (subunit)	Cofactor preference	Vmax for l-xylulose (U/mg)	Km for l-xylulose (mM)	Km for NADPH (mM)	Vmax for xylitol (U/mg)	Km for xylitol (mM)	Coding gene (GenBank accession No.)	Identity^c (%)	Reference
Rhizomucor pusillus	RpLXR	98,000	28,341^a	NADPH	106	8.71	0.0095	253	301	Rplxr3 (LC081188)	–	This study
Aspergillus niger	AnLXR	250,000	32,000	NADPH	200	16.5	0.03	33	925	–	–	5)
Aspergillus niger	rAnLXR	–	32,044^a	NADPH	650	25	–	–	–	lxrA (JGI177736)	27	6)
Hypocrea jecorina	rHjMDH		28,428^a	NADPH	4.50	16	–	–	–	lxr1 (AF375616)	42	7,8)
Hypocrea jecorina	rHjLXR	–	30,563^a	NADPH	367	16	0.13	–	–	lxr3 (BK008567)	41	9)
Ambrosiozyma monospora	rAmLXR	–	29,495^a	NADH	102^b	9.6^b	N.D.	37.8^b	7.2^b	ALX1 (AJ583159)	34	16)

^a Molecular weights were calculated from the deduced amino acid sequences.

^b LXR activities were measured with NAD(H) as a cofactor.

^c Each E-value was calculated with the amino acid sequence of RpLXR using the BLAST program.

Fig. 3. Phylogenetic tree of l-xylulose reductase from R. pusillus NBRC 4578 and its homologous proteins. The phylogenetic tree was constructed using ClustalW and MEGA7. The arrow showed RpLXR sequence determined in this study.

Fig. 4. Expression of the Rplxr3 gene in R. pusillus NBRC 4578 cultured on d-glucose (▼), l-xylose (●), l-arabinose (○), d-mannose (□), and glycerol (◊). The expression level of mycelia cultivated for 24 h on L-arabinose set as 1.0.

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