Molecular cloning and characterization of Brugia malayi hexokinase.
5' EST from filarial gene database has been subjected to 3' rapid amplification of cDNA ends (RACE), semi-nested PCR and PCR to obtain full-length cDNA of Brugia malayi. Full-length hexokinase gene was obtained from cDNA using gene specific primers. The elicited PCR product was cloned, sequenced and expressed as an active enzyme in Escherichia coli. Sequence analysis of B. malayi hexokinase (BmHk) revealed 59% identity with nematode Caenorhabditis elegans but low similarity with all other available hexokinases including human. BmHk, an apparent tetramer with subunit molecular mass of 72 kDa, was able to phosphorylate glucose, fructose, mannose, maltose and galactose. The Km values for glucose, fructose and ATP were found to be 0.035+/-0.005, 75+/-0.3 and 1.09+/-0.5 mM respectively. BmHk was strongly inhibited by ADP, glucosamine, N-acetyl glucosamine and mannoheptulose. The recombinant enzyme was found to be activated by glucose-6-phosphate. ADP exhibited noncompetitive inhibition with the substrate glucose (Ki=0.55 mM) while, mixed type of inhibition was observed with inorganic pyrophosphate (PPi) when ATP was used as substrate (Ki=9.92 microM). The enzyme activity is highly dependent on maintenance of free sulfhydryl groups. CD analysis indicated that BmHk is composed of 37% alpha-helices and 26% beta-sheets. The observed differences in kinetic properties of BmHk as compared to host enzyme may facilitate designing of specific inhibitors against BmHk.
Singh
Alok Ranjan
AR
Division of Biochemistry, Central Drug Research Institute, Lucknow, India.
Joshi
Shweta
S
Arya
Rahul
R
Kayastha
Arvind Mohan
AM
Srivastava
Kishore Kumar
KK
Tripathi
Lalit Mohan
LM
Saxena
Jitendra Kumar
JK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017178:Brugia malayi / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D005368:Filariasis / Q000469:parasitology; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D006593:Hexokinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D051189:Murinae; D017422:Sequence Analysis, DNA
D015801:Helminth Proteins; D006593:Hexokinase