Glycine oxidase from Bacillus subtilis: Role of Histidine 244 and Methionine 261 * Angelo Boselli, Elena Rosini, Giorgia Letizia Marcone, Silvia Sacchi, Laura Motteran, Mirella S. Pilone, Loredano Pollegioni * , Gianluca Molla Department of Biotechnology and Molecular Sciences, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy Received 9 March 2007; accepted 27 April 2007 Available online 8 May 2007 Abstract The reactions of several mutants at position 244 and 261 of bacterial glycine oxidase (GO) were studied by stopped-flow and steady-state kinetic methods. Substituting H244 with phenylalanine, glutamate, and glutamine and M261 with histidine and tyrosine did not affect the ex- pression of GO and the physicochemical properties of bound FAD. All the H244 and M261 mutants of GO we prepared retained activity in both steady-state and stopped-flow kinetic studies, indicating they do not serve as key elements in glycine and sarcosine oxidation. We demonstrated that the substitution of H244 significantly affected the rate of flavin reduction with glycine even if this change did not modify the turnover num- ber, which is frequently increased compared to wild-type GO. However, substitution of M261 affected the interaction with substrates/inhibitors and the rate of flavin reduction with sarcosine and resulted in a decrease in turnover number and efficiency with all the substrates tested. The considerable decrease in the rate of flavin reduction changed the conditions such that it was partially rate-limiting in the catalytic cycle compared to the wild-type GO. Our studies show some similarities, but also major differences, in the catalytic mechanism of GO and other flavooxidases also active on glycine and sarcosine and give insight into the mode of modulation of catalysis and substrate specificities. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Flavoprotein; Site-directed mutagenesis; Substrate recognition; Thiamine biosynthesis 1. Introduction Glycine oxidase (GO, EC 1.4.3.19) is a flavoprotein consisting of four identical subunits (369 residues each) and containing one molecule of non-covalently bound FAD per 42-kDa protein molecule [1,2]. It catalyzes the oxidative de- amination of various amines, preferentially those of a small size (e.g., glycine and sarcosine), and partially shares the sub- strate specificity with both D-amino acid oxidase (DAAO) and sarcosine oxidase (SOX) (for recent reviews on the mechanism of amine oxidation by flavoproteins see refs. [3,4]). The free-form crystal structure of B. subtilis GO was solved initially at 2.3 A ˚ resolution [5] and subsequently as a complex with the inhibitor glycolate at 1.8 A ˚ resolution [6]. From a structural point of view, GO is classified as a member of the glutathione reductase family (subfamily GR 2 ): dimethyl- glycine oxidase, monomeric sarcosine oxidase (MSOX), and DAAO represent closer structural neighbors; however, a lim- ited sequence identity is evident, for example, 18.4% of sequence identity between GO and DAAO [7]. SOX catalyzes the oxidative demethylation of sarcosine (N-methylglycine) to form glycine and formaldehyde, and DAAO catalyzes the oxidative deamination of neutral and (with a lower efficiency) basic D-amino acids to give the corresponding a-keto acids and ammonia. In both cases, the reduced coenzyme is reoxidized by molecular oxygen to yield H 2 O 2 . Although DAAO and SOX show a wide similarity in substrate specificity, they do not efficiently oxidize glycine. Previous investigations of the * Enzymes: Glycine oxidase (GO, EC 1.4.3.19), D-amino acid oxidase (DAAO, EC 1.4.3.3), Sarcosine oxidase (SOX, EC 1.5.3.1), MSOX, mono- meric sarcosine oxidase. * Corresponding author. Tel.: þ39 0332 421 506; fax: þ39 0332 421 500. E-mail address: loredano.pollegioni@uninsubria.it (L. Pollegioni). 0300-9084/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2007.04.019 Biochimie 89 (2007) 1372e1380 www.elsevier.com/locate/biochi