PLoS One. 2014 Oct 15;9(10):e110054.

Human valacyclovir hydrolase/biphenyl hydrolase-like protein is a highly efficient homocysteine thiolactonase

Judit Marsillach1,2, Stephanie M. Suzuki1, Rebecca J. Richter1, Matthew G. McDonald3, Peter M. Rademacher3, Michael J. MacCoss2, Edward J. Hsieh2, Allan E. Rettie3, Clement E. Furlong1,2

Departments of 1Medicine (Division of Medical Genetics), 2Genome Sciences and 3Medicinal Chemistry, University of Washington, Seattle, Washington, United States.



Homocysteinylation of lysine residues by homocysteine thiolactone (HCTL), a reactive homocysteine metabolite, results in protein aggregation and malfunction, and is a well-known risk factor for cardiovascular, autoimmune and neurological diseases. Human plasma paraoxonase-1 (PON1) and bleomycin hydrolase (Blmh) have been reported as the physiological HCTL detoxifying enzymes. However, the catalytic efficiency of HCTL hydrolysis by Blmh is low and not saturated at 20 mM HCTL. The catalytic efficiency of PON1 for HCTL hydrolysis is 100-fold lower than that of Blmh. A homocysteine thiolactonase (HCTLase) was purified from human liver and identified by mass spectrometry (MS) as the previously described human biphenyl hydrolase-like protein (BPHL). To further characterize this newly described HCTLase activity, BPHL was expressed in Escherichia coli and purified. The sequence of the recombinant BPHL (rBPHL) and hydrolytic products of the substrates HCTL and valacyclovir were verified by MS. We found that the catalytic efficiency (kcat/Km) of rBPHL for HCTL hydrolysis was 7.7 x 104 M-1s-1, orders of magnitude higher than that of PON1 or Blmh, indicating a more significant physiological role for BPHL in detoxifying HCTL.

PMID: 25333274; PMCID: PMC4198189



Plasma levels of homocysteine (Hcy) are a major vascular risk factor. It has been known for many years that subjects with cardiovascular disease and a history of stroke have an increased risk of developing Alzheimer’s disease (AD) [1]. Thus, it is reasonable to hypothesize that high levels of Hcy could also be a risk factor for AD. In fact, a few years ago it was demonstrated that indeed hyperhomocysteinemia is a risk factor not only for AD but also for other forms of dementia [2-3]. Despite extensive research in this topic, the mechanism by which hyperhomocysteinemia leads to cardiovascular disease and/or AD is not known. What it is known is that a reactive metabolite from Hcy, homocysteine thiolactone (HCTL), which is produced through an error-editing process by methionyl tRNA-synthetase can modify proteins in vivo altering their physiological function and resulting in the development of diseases [4-5].

HCTL has been reported to be hydrolyzed by enzymes with homocysteine thiolactonase activity, such as serum paraoxonase-1 (PON1) [6] and intracellular bleomycin hydrolase (Blmh) [7]. However, the catalytic efficiency of these enzymes for HCTL is quite low. Using conventional chromatography, mass spectrometry and microbial expression of recombinant protein, our group has characterized a more efficient homocysteine thiolactonase previously identified as biphenyl hydrolase-like protein (BPHL), identifying an important physiological role for this enzyme.

Human studies on the therapeutic benefit of Hcy reduction, such as folic acid and vitamin B supplementation, have reported negative conclusions. Therefore, research focusing on the mechanisms for detoxifying HCTL is important. Our recent study published in PLoS One sheds light into these needs by proposing BPHL as a physiologically relevant homocysteine thiolactonase that should be further studied in vivo. BPHL is highly expressed in liver and kidney, organs where Hcy and HCTL are mostly detoxified, but is also found in many brain areas, particularly in cortex and hippocampus which are the most affected areas in AD. With the hypothesis that BPHL plays an important role in detoxifying HCTL in vivo and therefore preventing development of diseases such as AD, our future efforts will involve characterization of the physiological significance of BPHL’s homocysteine thiolactonase activity and investigation of any genetic variability of BPHL that could predispose individuals to AD.

JM fig1

Figure 1. Potential mechanism of the development of neurological and cardiovascular diseases by high homocysteine levels. BPHL would prevent homocysteine thiolactone accumulation



JM fig2

Figure 2. Homocysteinylation of proteins. HCTL incorporates Hcy in lysine residues of proteins. The resulting Hcy adduct affects protein stability and function.



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Dr. Clement Furlong and Dr. Judit Marsillach, Division of Medical Genetics, Box 357720, University of Washington, 98195-7720 Seattle, Washington, United States of America; Phone: +1 2065431193; E-mail:,



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