Coronary artery disease is the most common cause of death globally and is linked to a number of risk factors including serum low density lipoprotein, high density lipoprotein, triglycerides and lipoprotein(a). Recently two proteins, angiopoietin-like protein 3 and 4, have emerged from genetic studies as being factors that significantly modulate plasma triglyceride levels and coronary artery disease. The exact function and mechanism of action of both proteins remains to be elucidated, however, mutations in these proteins results in up to 34% reduction in coronary artery disease and inhibition of function results in reduced plasma triglyceride levels. Here we report the crystal structures of the fibrinogen-like domains of both proteins. These structures offer new insights into the reported loss of function mutations, the mechanisms of action of the proteins and open up the possibility for the rational design of low molecular weight inhibitors for intervention in coronary artery disease.

Human aldehyde oxidase ( hAOX 1) is a molybdenum enzyme with high toxicological importance, but its physiological role is still unknown. hAOX 1 metabolizes different classes of xenobiotics and is one of the main drug‐metabolizing enzymes in the liver, along with cytochrome P450. hAOX 1 oxidizes and inactivates a large number of drug molecules and has been responsible for the failure of several phase I clinical trials. The interindividual variability of drug‐metabolizing enzymes caused by single nucleotide polymorphisms ( SNP s) is highly relevant in pharmaceutical treatments. In this study, we present the crystal structure of the inactive variant G1269R, revealing the first structure of a molybdenum cofactor (Moco)‐free form of hAOX 1. These data allowed to model, for the first time, the flexible Gate 1 that controls access to the active site. Furthermore, we inspected the thermostability of wild‐type hAOX 1 and hAOX 1 with various SNP s (L438V, R1231H, G1269R or S1271L) by CD spectroscopy and Thermo FAD , revealing that amino acid exchanges close to the Moco site can impact protein stability up to 10 °C. These results correlated with biochemical and structural data and enhance our understanding of hAOX 1 and the effect of SNP s in the gene encoding this enzyme in the human population. Enzymes Aldehyde oxidase ( EC1.2.3.1 ); xanthine dehydrogenase ( EC1.17.1.4 ); xanthine oxidase ( EC1.1.3.2 ). Databases Structural data are available in the Protein Data Bank under the accession number 6Q6Q .

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Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitorsthioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug−drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

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Human aldehyde oxidase (hAOX1) is a molybdoflavoenzyme that belongs to the xanthine oxidase (XO) family. hAOX1 is involved in phase I drug metabolism, but its physiological role is not fully understood to date and preclinical studies consistently underestimated hAOX1 clearance. In the present work, we report an unexpected effect of the common sulfhydrylcontaining reducing agents, e.g. dithiothreitol (DTT), on the activity of hAOX1 and mouse

Human aldehyde oxidase (hAOX1) is a molybdoenzyme that oxidizes aldehydes and N‐heterocyclic compounds, thereby generating hydrogen peroxide (H2O2) and superoxide during turnover. hAOX1 has been shown previously to be inactivated under turnover conditions by H2O2. Here, we investigated the effect of exogenously added H2O2 on the activity of hAOX1. We show that exogenously added H2O2 did not affect the enzyme activity under aerobic conditions, but completely inactivated the enzyme under anaerobic conditions. We propose that this effect is based on the reducing power of H2O2 and the susceptibility of the reduced molybdenum cofactor (Moco) to lose the sulfido ligand. When oxygen is present, the enzyme is rapidly reoxidized. We believe that our study is significant in understanding the detailed effect of reactive oxygen species on the inactivation of hAOX1 and other molybdoenzymes.

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