Crystal structures of Babesia microti lactate dehydrogenase BmLDH reveal a critical role for Arg99 in catalysis

Abstract

The tick-and transfusion-transmitted human pathogen Babesia microti infects host erythrocytes to cause the pathologic symptoms associated with human babesiosis, an emerging disease with worldwide distribution and potentially fatal clinical outcome. Drugs currently recommended for the treatment of babesiosis are associated with a high failure rate and significant adverse events, highlighting the urgent need for more-effective and safer babesiosis therapies. Unlike other apicomplexan parasites, B. microti lacks a canonical lactate dehydrogenase (LDH) but instead expresses a unique enzyme, B. microti LDH (BmLDH), acquired through evolution by horizontal transfer from a mammalian host. Here, we report the crystal structures of BmLDH in apo state and ternary complex (enzyme-NADH-oxamate) solved at 2.79 and 1.89Å. Analysis of these structures reveals that upon binding to the coenzyme and substrate, the active pocket of BmLDH undergoes a major conformational change from an opened and disordered to a closed and stabilized state. Biochemical assays using wild-type and mutant B. microti and human LDHs identified Arg99 as a critical residue for the catalytic activity of BmLDH but not its human counterpart. Interestingly, mutation of Arg99 to Ala had no impact on the overall structure and affinity of BmLDH to NADH but dramatically altered the closure of the enzyme's active pocket. Together, these structural and biochemical data highlight significant differences between B. microti and human LDH enzymes and suggest that BmLDH could be a suitable target for the development of selective antibabesial inhibitors.

Identifying the Naphthalene-Based Compound 3,5-Dihydroxy 2-Napthoic Acid as a Novel Lead Compound for Designing Lactate Dehydrogenase-Specific Antibabesial Drug

“…Our previous study based on the crystal structures of BmLDH indicated that the mutation of residue Arg99 to Ala significantly reduced the enzyme activity of BmLDH (up to 86%), but had no impact on Human LDH-A (Yu et al, 2019). For testing the probability of BmLDH as a selective drug target, the potent inhibitors interacting with the residue Arg99 were screened via a molecular docking based on the crystal structure of BmLDH from a series of gossypol derivatives or structural analogs.…”

“…In Babesia bovis, gossypol inhibited BbLDH enzymatic activity to cause the death of B. bovis and its K i and IC 50 in vitro values were determined as 0.085 and 50 mM (Bork et al, 2004). In B. microti, the catalytic activity of BmLDH was inhibited by gossypol at 0.67 mM (K i ), and the IC 50 value of gossypol against B. microti in vitro was 7.07 mM (Yu et al, 2019). Previous studies showed that these derivatives of gossypol exhibited obviously lower cytotoxicity toward mammalian cells than the parent compound (Royer et al, 1991;Royer et al, 1995).…”

“…Interestingly, the enzyme of B. microti (BmLDH) acquired by a lateral gene transfer event has been identified as a mammalianlike LDH enzyme, and is significantly different from all the other protozoans in the unusual event (Cornillot et al, 2012;Silva et al, 2016). Our previous study with the crystal structures of BmLDH indicated that the enzyme activity of BmLDH could be dramatically inhibited by gossypol and oxamate, and the residue Arg99 was crucial in the catalysis of BmLDH, but not in Human LDH-A (Yu et al, 2019). All the available reports indicate that the BmLDH could serve as a novel drug target for the development of new strategies to treat the human parasitosis.…”

Kinetic Characterization and Inhibitor Screening of Pyruvate Kinase I From Babesia microti

“…To determine the enzymatic activity of purified rBmPYKI, the GST tag was removed from the recombinant protein; and the indirect coupled lactate dehydrogenase (LDH) assay was applied. Recombinant B. microti LDH (rBmLDH) protein, the only one isoform in B. microti , was expressed and purified according to previously published protocol ( Yu et al, 2019 ). The standard enzyme kinetic assay was conducted by monitoring the decrease in NADH at a wavelength of 340 nm using a microplate reader (BioTek, Winooski, VT, United States).…”

The Structural Basis of Babesia orientalis Lactate Dehydrogenase

“…In B. bovis , phenolic aldehyde gossypol dramatically inhibited the catalysis activity of BbLDH with a K i value of 0.85 μM, and the value was lower than human LDH-A (HuLDH-A) (1.9 μM) and CpLDH (14.9 μM)), and similar to PfLDH (0.7 μM) and BmLDH (0.67 μM). Interestingly, gossypol treatment also reduced the in vitro growth of these parasites with IC 50 values of 50 μM for B. bovi s, 15.3 μM for P. falciparum , 11.8 μM for C. parvum , and 7.07 μM for B. microti ( Yu et al., 2001 ; Bork et al., 2004 ; Choi et al., 2007b ; Zhang et al., 2015 ; Yu et al., 2019 ). These reports suggest that LDHs of apicomplexan parasites might act as a future drug candidate for finding new anti-parasitic drugs.…”