Elsevier

Archives of Biochemistry and Biophysics

Volume 564, 15 December 2014, Pages 156-163
Archives of Biochemistry and Biophysics

A pre-steady state and steady state kinetic analysis of the N-ribosyl hydrolase activity of hCD157

https://doi.org/10.1016/j.abb.2014.09.008Get rights and content

Highlights

  • Nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are hydrolyzed by hCD157.

  • The enzyme hydrolyzes NR with a kcat/KM of 17 μM−1 s−1. NR is the preferred substrate for hCD157.

  • The long half-life (7 s) of the catalytic intermediate suggests that hydrolysis proceeds through a covalent intermediate.

Abstract

hCD157 catalyzes the hydrolysis of nicotinamide riboside (NR) and nicotinic acid riboside (NAR). The release of nicotinamide or nicotinic acid from NR or NAR was confirmed by spectrophotometric, HPLC and NMR analyses. hCD157 is inactivated by a mechanism-based inhibitor, 2′-deoxy-2′-fluoro-nicotinamide arabinoside (fNR). Modification of the enzyme during the catalytic cycle by NR, NAR, or fNR increased the intrinsic protein fluorescence by approximately 50%. Pre-steady state and steady state data were used to derive a minimal kinetic scheme for the hydrolysis of NR. After initial complex formation a reversible step (360 and 30 s−1) is followed by a slow irreversible step (0.1 s−1) that defined the rate limiting step, or kcat. The calculated KMapp value for NR in the hydrolytic reaction is 6 nM. The values of the kinetic constants suggest that one biological function of cell-surface hCD157 is to bind and slowly hydrolyze NR, possibly converting it to a ligand-activated receptor. Differences in substrate preference between hCD157 and hCD38 were rationalized through a comparison of the crystal structures of the two proteins. This comparison identified several residues in hCD157 (F108 and F173) that can potentially hinder the binding of dinucleotide substrates (NAD+).

Introduction

Nicotinamide adenine dinucleotide (NAD+)2 is a cofactor in over 400 oxidation–reduction reactions. NAD+ is also used in protein poly/mono ADP-ribosylation, protein deacetylation, transcriptional repression, vitamin biosynthesis and gene splicing reactions [1], [2], [3], [4], [5]. Pharmacological stimulation of NAD+ synthesis or inhibition of NAD+ degradation has therapeutic promise for the treatment of a number of human conditions and diseases in which NAD+ levels are decreased relative to the normal state [6], [7], [8], [9], [10], [11]. For example, perturbed NAD+ metabolism occurs in pellagra, which is easily cured by niacin supplementation [12]. Successful pharmacological intervention in the disease state is dependent upon a fundamental understanding of the anabolic and catabolic enzymes involved in producing the NAD+ deficit.

Aplysia ADP-ribosyl cyclase (ARC) is the first member of a family of NAD+ metabolizing enzymes (E.C.3.2.2.5) to be characterized [13]. The enzyme catalyzes the formation of nicotinamide (Nam) and the calcium mobilization hormone cyclic adenosine diphosphate ribose (cADPR) from NAD+, with retention of the β-stereochemistry at the C1′-ribose residue. The mammalian paralog, CD38, is primarily a hydrolytic enzyme of the pyridine nucleotides, NAD+, NADP+ and NMN. Using NAD+ as substrate CD38 catalyzes the formation of approximately 1% cADPR and 99% ADPR and Nam. These enzymes also efficiently catalyze selected base-exchange reactions with Nam analogs (and pyridine nucleotides) that has been used to characterize and isolate alternative substrates and inhibitors of these enzymes [14], [15], [16], [17].

CD157 (often referred to as; bone marrow stromal antigen BST-1, NAD+ nucleosidase or cyclic ADP-ribose hydrolase 2) was initially cloned by differential screening of synovial fluid from patients with a severe form of rheumatoid arthritis [18], [19]. The gene for this enzyme is located on human chromosome 4 (4p15.33) and is a product of gene duplication of the CD38 locus [20]. The exon structure and gene regulatory elements are highly conserved between CD38 and CD157. Because hCD157 is a younger gene than hCD38, the human protein is 78–89% identical to the mouse and rat homolog, respectively. Despite their common ancestry there are notable differences. CD38 is an integral membrane protein, whereas CD157 is associated with the cellular membrane through a C-terminal glycophosphatidylinositol (GPI) linkage [20]. CD38 is a powerful hydrolytic catalyst (NAD+ kcat/KM value of 10 μM−1 s−1), whereas CD157 is an ineffective (NAD+ and NGD+ kcat/KM value of 0.2–1 mM−1 s−1) hydrolytic catalyst [15], [21], [22]. In summary CD157 is a protein with novel, but related biochemical properties.

Nicotinamide riboside (NR) was initially characterized in the 1940s and was recently shown to function as a precursor vitamin for eukaryotic NAD+ biosynthesis [23], [24]. Intracellular phosphorylation of NR produces NMN, an intermediate that is then subsequently adenylated to form NAD+ in the NAD+ salvage pathway. Because exogenously-added NR raises intracellular NAD+ levels in mammalian cells (2–3-fold) NR could be a dietary supplement that elevates NAD levels [25]. It is possible that inhibition of NR catabolism may be a potential mechanism for increasing cellular NAD+ levels.

During the course of characterizing the biochemical selectivity of CD38 inhibitors, we discovered that hCD157 prefers NR over NAD+ as a substrate for hydrolysis (kcat/KM value of 17 μM−1 s−1). Herein, we present evidence that the enzyme uses covalent catalysis with formation of a ribosylated enzyme intermediate. In the absence of nicotinamide, the level of ribosylated enzyme is high at low levels of NR. Thus the enzyme is effectively capturing the ribose portion of NR at very low levels of NR. The proposed ribosylated intermediate is relatively long-lived, suggesting that NR may function to activate hCD157 as a receptor. Alternative substrates (analogs of NR) or antagonists (inhibitors) of hydrolytic function may prove to be useful therapeutic agents for the treatment of a number of human diseases.

Section snippets

Materials

NAD+, NMN, nicotinamide, alkaline phosphatase, phosphate buffered saline (PBS), Hepes, nicotinic acid, and EDTA were from Sigma Chemical Co. hCD157 (catalog number 4736-AC, lot # RLZ0311071) was purchased from R & D Systems, Minneapolis, MN or from Reprokine Ltd., Rehovot, Israel (catalog number RKQ 10588). NR was purchased from Toronto Research Chemicals.

Purification of recombinant proteins

Our initial characterization of two commercially available hCD157 proteins revealed biochemical differences between the proteins. Due to

Steady-state hydrolysis of NR

Time-dependent changes in the UV properties of NR in the Standard Buffer were observed upon addition of hCD157. Similar changes were observed in 50 mM Hepes pH 7.0, 1 mM EDTA. The reaction was not dependent on phosphate or on pyrophosphate. The UV difference spectrum of the spent solution with enzyme vs a solution lacking enzyme revealed a maximal absorbance change at 262 nm. The normalized difference spectrum was identical to that predicted from the hydrolysis of NR. Using an extinction

Discussion

CD157 or BST-1 (NAD+ nucleosidase, cyclic ADP-ribose hydrolase 2) arose approximately 500 million years ago through gene duplication of CD38 and today has only 36% sequence identity with CD38. Initial biochemical characterization determined that hCD157 is a weak NAD+ hydrolase and ADP-ribosyl cyclase (kcat/KM value for NAD of 200 M−1 s−1). Our experiments have shown that this enzyme has evolved to recognize, bind and hydrolyze NR with a kcat/KM value of 17 μM−1 s−1. The exquisite

Acknowledgments

The authors would like to thank Curt Haffner and David Becherer for their support in characterizing CD38 inhibitors. Mark Bickett for initial preparations of NR; Yingnian Shen for cloning hCD157 into GSK Bacmam expression vectors; Scott Sigethy for preparing Bacmam virus stocks; George Barrett for large scale cell culture; Mary B. Moyer for LC/MS peptide sequencing of fNR-modified hCD157; Ginger Tomberlin for HPLC analyses; Timothy Spitzer for NMR analyses; Richard Caldwell and Bruce

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