Structure and activity of the axon guidance protein MICAL

Abstract

During development, neurons are guided to their targets by shortand long-range attractive and repulsive cues. MICAL, a large multidomain protein, is required for the combined action of semaphorins and plexins in axon guidance. Here, we present the structure of the N-terminal region of MICAL (MICAL fd) determined by x-ray diffraction to 2.0 Å resolution. The structure shows that MICAL fd is an FAD-containing module structurally similar to aromatic hydroxylases and amine oxidases. In addition, we present biochemical data that show that MICAL fd is a flavoenzyme that in the presence of NADPH reduces molecular oxygen to H 2O2 (Km,NAPDH ‫؍‬ 222 M; kcat ‫؍‬ 77 sec ؊1 ), a molecule with known signaling properties. We propose that the H 2O2 produced by this reaction may be one of the signaling molecules involved in axon guidance by MICAL.hydrogen peroxide ͉ hydroxylase ͉ monooxygenase ͉ x-ray diffraction

Release of MICAL Autoinhibition by Semaphorin-Plexin Signaling Promotes Interaction with Collapsin Response Mediator Protein

“…The crystal structure of the monooxygenase (MO) domain of mouse MICAL1 reveals that it shares a structural homology to aromatic hydroxylases, such as p-hydroxybenzoate hydroxylase (pHBH) from Pseudomonas fluorescens (Nadella et al, 2005;Siebold et al, 2005), and has detectable enzymatic activity (Nadella et al, 2005). MICAL is an NADPH-dependent enzyme, consistent with monooxygenase function (Massey, 1995).…”

“…The Amplex Red Hydrogen Peroxide/Peroxidase Assay kit (Invitrogen) has been used to measure hydrogen peroxide production from the purified enzymatic domain of MICAL1 (Nadella et al, 2005). We further adapted the kit to study the activity of MICAL1 truncation mutants in HEK293T cell lysates.…”

“…Indeed, it has been shown that MICAL is an NADPH-dependent enzyme (Nadella et al, 2005;Siebold et al, 2005). A previous report demonstrated the use of an enzyme-linked assay to measure the activity of purified MICAL FAD-binding monooxygenase domain (amino acids 1-489) based on hydrogen peroxide production (Nadella et al, 2005). We adapted this assay to test the activity of MICAL truncation mutants in lysates of transfected HEK293T cells.…”

“…The reduced form of the flavoenzyme reacts with molecular oxygen to form a detectable flavin C(4a) hydroperoxide intermediate, which in the absence of substrate, decomposes to produce hydrogen peroxide and reoxidized FAD (Massey, 1994(Massey, , 1995. Indeed, it has been shown that MICAL is an NADPH-dependent enzyme (Nadella et al, 2005;Siebold et al, 2005). A previous report demonstrated the use of an enzyme-linked assay to measure the activity of purified MICAL FAD-binding monooxygenase domain (amino acids 1-489) based on hydrogen peroxide production (Nadella et al, 2005).…”

“…However, the FAD-binding domain alone, tMICAL 1-450, was expressed at lower levels, so its direct interaction cannot be compared directly. Because it is thought that mouse MICAL1 forms a globular protein (Nadella et al, 2005), multiple residues located throughout the region spanning the FAD and LIM domains may mediate CRMP binding rather than a specific linear chain of amino acids.…”