Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase

Table 1

Primers used for site mutation.

Plasmid	Primer^a	Sequence (5′~3′)^b	Purpose	Mutagenesis amino acid (codon)
pMDH	MDH-F	CGCGGATCC ^c ATGGCACGCAACAAGATTG	Gene cloning
	MDH-R	CGCGTCGAC ^d TTATTTCAGCGACGGAGCA	Gene cloning
pMDH-R89L	MDH-R89LF	GGCATGAGCCTCGACGATCTCCTGGGC	Site 89 Arg mutation	Arg(CGC) → Leu(CUC)
	MDH-R89LR	GCCCAGGAGATCGTCGAGGCTCATGCC	Site 89 Arg mutation	Arg(CGC) → Leu(CUC)
pMDH-D149V	MDH-D149VF	GGC GTT CTC GTC AGC GCC CGCTTCCGT	Site 149 Asp mutation	Asp(GAC) → Val(GUC)
	MDH-D149VR	ACG GAA GCG GGCGCTGACGAGAACGCC	Site 149 Asp mutation	Asp(GAC) → Val(GUC)
pMDH-R152L	MDH-R152LF	GACAGCGCCCTCTTCCGTTATTTCCTC	Site 152 Arg mutation	Arg(CGC) → Leu(CUC)
	MDH-R152LR	GAGGAAATAACGGAAGAGGGCGCTGTC	Site 152 Arg mutation	Arg(CGC) → Leu(CUC)
pMDH-H176P	MDH-H176PF	CTGGGTGGCCCCGGCGATTCGATGGTT	Site176 His mutation	His(CAC) → Pro(CCC)
	MDH-H176PR	AACCATCGAATCGCCGGGGCCACCCAG	Site 176 His mutation	His(CAC) → Pro(CCC)
pMDH-D178V	MDH-D178VF	GGCCACGGCGTTTCGATGGTTCCGCTG	Site 178 Asp mutation	Asp(GAU) → Val(GUU)
	MDH-D178VR	CAGCGGAACCATCGAAACGCCGTGGCC	Site 178 Asp mutation	Asp(GAU) → Val(GUU)
pMDH-A231V	MDH-A231VF	ACCGGCTCGGTTTTCTACGCTCCGGCG	Site 231 Ala mutation	Ala(GCU) → Val(GUU)
	MDH-A231VR	CGCCGGAGCGTAGAAAACCGAGCCGGT	Site 231 Ala mutation	Ala(GCU) → Val(GUU)

aAmino acids are represented by their one-letter abbreviation, and the number indicates the localization of the mutated residue in the amino acid sequence of MDH.
^bMutations that have been introduced in the oligonucleotides of the mdh gene are indicated in bold.
^c,dBamHI and SalI sites were underlined, respectively.