Protein Engineering of the 4-Methyl-5-Nitrocatechol Monooxygenase from Burkholderia sp. Strain DNT for Enhanced Degradation of Nitroaromatics

Thammajun Leungsakul; Glenn R. Johnson; Thomas K. Wood

doi:10.1128/AEM.02966-05

DOI: 10.1128/AEM.02966-05

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FIG. 1.
Oxidation of 4M5NC, 4NC, 4NP, and 3M4NP by wild-type DntB and variant M22L/L380I. The proposed oxidation pathways of 4NC, 4NP, and 3M4NP by the DntB M22L/L380I variant are marked with asterisks. Oxidation of 4M5NC by wild-type DntB is based on Haigler et al. (11, 12). The retention time and maximum absorbance wavelength used in the HPLC analysis were 14.93 min and 245.3/353.1 nm for 4M5NC, 8.31 min and 264.4 nm for 2,4,5-trihydroxytoluene or 2-hydroxy-5-methylquinone, 12.0 min and 242.9/345.9 nm for 4NC, 4.6 min and 253.6 nm for 1,2,4-trihydroxybenzene, 15.4 min and 225.1/315.8 nm for 4NP, 3.8 min and 221.6/287.1 nm for hydroquinone, 19.1 min and 312.2 nm for 3M4NP, and 6.08 min and 288 nm for methylhydroquinone, respectively.

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TABLE 1.

PCR primers used for constructing pBS(Kan)DntB DNT, for saturation mutagenesis of positions M22 and L380 of DntB, and for sequence analysis of the wild-type dntB and its variants

Purpose	Primer	Sequence^a
Plasmid construction	DntB DNT front	5′-GCCAGGGTTTCGGCCCTAGAATTCCGTCATCCC-3′
	DntB DNT rear	5′-CGGCGGGCGTCTAGAGTGCTCCGCTCGGC-3′
Error-prone PCR	DntAa NotI Front	5′-AGTGAGCGCAAGCGGCCGCATGTGAGTTAGCTCACTC-3′
	pBSKan Rev	5′-GGGCCTCTTCGCTATTACGCCAGC-3′
Saturation mutagenesis	dntB DNT M22 front	5′-CCTCATCGTGGGCGGGAGCNNSGTCGGACTGTCC-3′
	dntB DNT M22 rear	5′-GAACAACGCCGTGGACAGTCCGACSNNGCTCCCGCCC −3′
	dntB DNT L380 front	5′-CCTTCAGCCGCTACATCCGCCGGNNSGCACCCGAGTTCC −3′
	dntB DNT L380 rear	5′-GCTCGTCCAGGAACTCGGGTGCSNNCCGGCGGATGTAGC-3′
	DntBDNT M+L front	5′ GTCAGCGCCGGCTTCACTACCGACACCGCCGTCG 3′
	DntBDNT M+L rear	5′ CCGGACGAGTTCGACGGCGGTGTCGGTAGTGAAGC 3′
Sequence analysis	TpMO107FO	5′-GACCATGATTACGCCAAGCGCGC-3′
	DNT DNTB2	5′-GTCTCTCGCCGGTGGGGAAACGG-3′
	DNT DNTB3 new	5′-GCTTCACTACCGACACCGCCGTCG-3′
	DNTAcXba Rev	5′-AGGGTTTTCCCAGTCACG −3′
	SEQ PET Front upstream	5′-CATACCCACGCCGAAACAAG-3′

↵ a Restriction and mutation sites are underlined and boldface. S indicates nucleotide C or G.

TABLE 2.
Initial rates of nitrite release for substrates 4M5NC, 4NC, 4NP, and 3M4NP by purified wild-type DntB DNT and purified M22L/L380I
Substrate (300 μM) Rate (nmol/min/mg protein) Relative^a
Wild-type DntB M22L/L380I
4M5NC 41 ± 4 58 ± 2 1.4
4NC 31 ± 3 49 ± 1 1.6
4NP 4 ± 2 39 ± 6 9.8
3M4NP 4.4 ± 0.7 17 ± 4 3.9
↵ a Relative to wild-type DntB DNT.

TABLE 3.

Kinetic values for the purified wild-type DntB and M22L/L380I enzymes toward 4NPa

Enzyme	Apparent V_max (nmol/min/ mg protein)	Apparent K_m (μM)	Apparent k_cat (s⁻¹)	Apparent k_cat/ K_m (s⁻¹ M⁻¹)
Wild-type DntB	14 ± 3	350 ± 75	0.014 ± 0.004	40 ± 14
M22L/L380I	44 ± 2	100 ± 5	0.045 ± 0.003	450 ± 38

a At 300 μM of NADPH. The rates were determined from nitrite release at 50, 100, 200, 300, 500, and 1,000 μM 4NP concentrations based on a Michaelis-Menten model.

TABLE 4.

Initial rates of nitrite release for the substrate 4NP^a

Strain	Codon change	Rate (nmol/min/mg protein)	Relative^b
Wild-type DntB		0.2 ± 0.1	1.0
M22L	ATG to TTG	0.1 ± 0.1	0.5
L380I	CTC to ATC	0.2 ± 0.2	1.0
M22L/L380I	ATG to TTG/CTC to ATC	0.5 ± 0.4	2.5
M22L/L380M	ATG to TTG/CTC to ATG	0.1 ± 0.1	0.5
M22S/L380V	ATG to TCG/CTC to GTG	0.6 ± 0.6	3.0

↵ a At 500 μM by E. coli TG1 expressing wild-type DntB DNT and its variants M22L, L380I, M22L/L380I, M22L/L380M, and M22S/L380V.
↵ b Relative to wild-type DntB.