Molecular cloning and functional characterization of fumarases C in Neisseria species.
Fumarase is one of the key enzymes in the TCA cycle and has been implicated in virulence and survival of some microorganisms under suboptimal environmental conditions. In this study, the fumC genes that encode fumarase C (FUMCs) from Neisseria meningitidis, N. gonorrhoeae and N. subflava were identified by homology-based analysis, cloned by polymerase chain reactions and fully sequenced. The inferred primary sequence of neisserial FUMCs showed a high degree of conservation with 97.8-98.7% amino acid identity. However, phylogenetic analysis revealed that these neisserial FUMCs are divergent from class II fumarases found in other microorganisms, rat and human. The putative fumC genes were subcloned into the expression vector, pGEX-6P-1 and efficiently expressed in Esherichia coli BL21. The purified recombinant fusion proteins obtained by affinity chromatography demonstrated high catalytic activities (120-180 U/mg), thus authenticating the identities and functionalities of the cloned genes. Whether FUMC has any physiological relevance to the pathogenesisity of Neisseriae must await future gene disruption or mutagenesis studies.
Goh
Liuh Ling
LL
Department of Microbiology, Faculty of Medicine, National University of Singapore, MD4A, 5 Science Drive 2, 117597, Singapore.
Barkham
Timothy
T
Sim
Tiow Suan
TS
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005649:Fumarate Hydratase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009343:Neisseria / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C095842:fumarase C; D005649:Fumarate Hydratase