Actin stimulates reduction of the MICAL-2 monooxygenase domain.
MICALs are large, multidomain flavin-dependent monooxygenases that use redox chemistry to cause actin to depolymerize. Little enzymology has been reported for MICALs, and none has been reported for MICAL-2, an enzyme vital for the proliferation of prostate cancer. The monooxygenase domains of MICALs resemble aromatic hydroxylases, but their substrate is the sulfur of a methionine of actin. In order to determine how closely MICAL-2 conforms to the aromatic hydroxylase paradigm, we studied its reaction with NAD(P)H. The enzyme has a strong preference for NADPH over NADH caused by a large difference in binding NADPH. A comparison of the reduction kinetics using protio-NADPH and [4R-(2)H]-NADPH showed that MICAL-2 is specific for the proR hydride of NADPH, as evidenced by a 4.8-fold kinetic isotope effect. The reductive half-reaction of the MICAL-2 hydroxylase domain is stimulated by f-actin. In the absence of actin, NADPH reduces the flavin relatively slowly; actin speeds that reaction significantly. The separate monooxygenase domain of MICAL-2 has the classic regulatory behavior of flavin-dependent aromatic hydroxylases (Class A monooxygenases): slow reduction of the flavin when the substrate to be oxygenated is absent. This prevents the wasteful consumption of reduced pyridine nucleotide and the production of harmful H2O2. Our results show that this strategy is used by MICAL-2. Thus, our data suggest that MICAL-2 could regulate catalysis through the monooxygenase domain alone; control by interactions with other domains of MICAL in the full-length enzyme may not be needed.
Department of Biological Chemistry, University of Michigan Medical School , 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0606, United States.
Claudia A
CA
McDonald
Ying Yi
YY
Liu
Bruce A
BA
Palfey
D000199:Actins / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D007700:Kinetics; D051379:Mice; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry; D010084:Oxidation-Reduction; D013237:Stereoisomerism
D000199:Actins; D017931:DNA Primers; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D009243:NAD; C507541:Mical1 protein, mouse; D006899:Mixed Function Oxygenases