A novel Porphyromonas gingivalis enzyme: An atypical dipeptidyl peptidase III with an ARM repeat domain.

Porphyromonas gingivalis, an asaccharolytic Gram-negative oral anaerobe, is a major pathogen associated with adult periodontitis, a chronic infective disease that a significant percentage of the human population suffers from. It preferentially utilizes dipeptides as its carbon source, suggesting the importance of dipeptidyl peptidase (DPP) types of enzyme for its growth. Until now DPP IV, DPP5, 7 and 11 have been extensively investigated. Here, we report the characterization of DPP III using molecular biology, biochemical, biophysical and computational chemistry methods. In addition to the expected evolutionarily conserved regions of all DPP III family members, PgDPP III possesses a C-terminal extension containing an Armadillo (ARM) type fold similar to the AlkD family of bacterial DNA glycosylases, implicating it in alkylation repair functions. However, complementation assays in a DNA repair-deficient Escherichia coli strain indicated the absence of alkylation repair function for PgDPP III. Biochemical analyses of recombinant PgDPP III revealed activity similar to that of DPP III from Bacteroides thetaiotaomicron, and in the range between activities of human and yeast counterparts. However, the catalytic efficiency of the separately expressed DPP III domain is ~1000-fold weaker. The structure and dynamics of the ligand-free enzyme and its complex with two different diarginyl arylamide substrates was investigated using small angle X-ray scattering, homology modeling, MD simulations and hydrogen/deuterium exchange (HDX). The correlation between the experimental HDX and MD data improved with simulation time, suggesting that the DPP III domain adopts a semi-closed or closed form in solution, similar to that reported for human DPP III. The obtained results reveal an atypical DPP III with increased structural complexity: its superhelical C-terminal domain contributes to peptidase activity and influences DPP III interdomain dynamics. Overall, this research reveals multifunctionality of PgDPP III and opens direction for future research of DPP III family proteins.

Expression of domain III of the envelope protein from GP-78: a Japanese encephalitis virus.

Acute encephalitis caused by the Japanese encephalitis virus (JEV) represents a growing epidemic and is a cause for concern in Southeast Asia. JEV is transmitted to humans through the bite of the Culicine mosquito species. The virus genome comprising of an RNA strand also encodes the envelope protein (E) which surrounds the virus. The E protein aids in fusion of virus with the cellular membrane of the host cell with the help of three structurally distinct domains (DI, DII, DIII) that are connected by flexible hinge regions. Of these domains, DIII (JEV-DIII) has been reported to interact with the cellular membrane, aid viral entry and viral replication. Hence JEV-DIII has the potential to be an antigen that can provide immune protection to a JEV infection. In this study, we describe the cloning and expression of DIII of GP-78, a virulent strain of JEV prevalent in India. Our data clearly shows that JEV-DIII expressed from pVAC1 in HEK293T cells is membrane targeted. To our knowledge, this is the first demonstration of a recombinant construct that may block JEV entry into the cells and/or evoke specific antibodies against JEV. Future studies will reveal if our construct will elicit significant immune responses which will alleviate or ameliorate the pro-inflammatory responses induced by JEV.

Crystal structure of the Saccharomyces cerevisiae monoglyceride lipase Yju3p.

Monoglyceride lipases (MGLs) are a group of α/ß-hydrolases that catalyze the hydrolysis of monoglycerides (MGs) into free fatty acids and glycerol. This reaction serves different physiological functions, namely in the last step of phospholipid and triglyceride degradation, in mammalian endocannabinoid and arachidonic acid metabolism, and in detoxification processes in microbes. Previous crystal structures of MGLs from humans and bacteria revealed conformational plasticity in the cap region of this protein and gave insight into substrate binding. In this study, we present the structure of a MGL from Saccharomyces cerevisiae called Yju3p in its free form and in complex with a covalently bound substrate analog mimicking the tetrahedral intermediate of MG hydrolysis. These structures reveal a high conservation of the overall shape of the MGL cap region and also provide evidence for conformational changes in the cap of Yju3p. The complex structure reveals that, despite the high structural similarity, Yju3p seems to have an additional opening to the substrate binding pocket at a different position compared to human and bacterial MGL. Substrate specificities towards MGs with saturated and unsaturated alkyl chains of different lengths were tested and revealed highest activity towards MG containing a C18:1 fatty acid.