title: Crystallographic study to determine the substrate specificity of an L-serine-acetylating enzyme found in the D-cycloserine biosynthetic pathway.
abstract: DcsE, one of the enzymes found in the d-cycloserine biosynthetic pathway, displays a high sequence homology to l-homoserine O-acetyltransferase (HAT), but it prefers l-serine over l-homoserine as the substrate. To clarify the substrate specificity, in the present study we determined the crystal structure of DcsE at a 1.81-Å resolution, showing that the overall structure of DcsE is similar to that of HAT, whereas a turn region to form an oxyanion hole is obviously different between DcsE and HAT: in detail, the first and last residues in the turn of DcsE are Gly(52) and Pro(55), respectively, but those of HAT are Ala and Gly, respectively. In addition, more water molecules were laid on one side of the turn region of DcsE than on that of HAT, and a robust hydrogen-bonding network was formed only in DcsE. We created a HAT-like mutant of DcsE in which Gly(52) and Pro(55) were replaced by Ala and Gly, respectively, showing that the mutant acetylates l-homoserine but scarcely acetylates l-serine. The crystal structure of the mutant DcsE shows that the active site, including the turn and its surrounding waters, is similar to that of HAT. These findings suggest that a methyl group of the first residue in the turn of HAT plays a role in excluding the binding of l-serine to the substrate-binding pocket. In contrast, the side chain of the last residue in the turn of DcsE may need to form an extensive hydrogen-bonding network on the turn, which interferes with the binding of l-homoserine.
authors: Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors: Kosuke
authors: K
authors: Oda
authors: Yasuyuki
authors: Y
authors: Matoba
authors: Takanori
authors: T
authors: Kumagai
authors: Masafumi
authors: M
authors: Noda
authors: Masanori
authors: M
authors: Sugiyama
mesh_terms: D000123:Acetyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003523:Cycloserine / Q000096:biosynthesis*; D004268:DNA-Binding Proteins; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012694:Serine / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D014764:Viral Proteins
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C069215:MuB protein, Enterobacteria phage Mu; D014764:Viral Proteins; D012694:Serine; D003523:Cycloserine; D000123:Acetyltransferases; C021369:homoserine O-acetyltransferase
references: Acta Crystallogr D Biol Crystallogr. 1994 Sep 1;50(Pt 5):760-3
references: 15299374
references: Eur J Pharmacol. 2002 Feb 15;437(1-2):73-7
references: 11864642
references: Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
references: 9757107
references: Methods Enzymol. 1997;276:307-26
references: 27754618
references: J Biol Chem. 2004 Oct 29;279(44):46143-52
references: 15302885
references: Am J Hosp Pharm. 1976 Mar;33(3):275-83
references: 769544
references: Biochemistry. 2003 May 20;42(19):5775-83
references: 12741835
references: Antimicrob Agents Chemother. 2012 Jul;56(7):3682-9
references: 22547619
references: J Bacteriol. 1972 Jun;110(3):978-87
references: 4555420
references: J Antibiot (Tokyo). 2003 Sep;56(9):762-7
references: 14632285
references: Biochemistry. 2005 Oct 18;44(41):13560-6
references: 16216079
references: Antimicrob Agents Chemother. 2010 Mar;54(3):1132-9
references: 20086163
references: Biochemistry. 1964 Apr;3:471-80
references: 14188160
references: Biochemistry. 2005 Dec 6;44(48):15768-73
references: 16313180
references: J Biol Chem. 1966 Nov 10;241(21):4955-65
references: 5332668
references: Biochemistry. 2000 Jul 25;39(29):8556-64
references: 10913262
references: J Biol Chem. 2008 Nov 14;283(46):31567-74
references: 18799456
references: Curr Opin Struct Biol. 1999 Dec;9(6):732-7
references: 10607665
references: Protein Eng. 1992 Apr;5(3):197-211
references: 1409539
references: Arch Biochem Biophys. 2005 Jan 1;433(1):85-95
references: 15581568
references: Antimicrob Agents Chemother. 2011 Jan;55(1):291-301
references: 20956591
references: Biochem Biophys Res Commun. 2007 Nov 30;363(4):1050-6
references: 17927957
references: Behav Brain Res. 2002 Feb 1;129(1-2):211-6
references: 11809513
references: Biochem Biophys Res Commun. 1971 Nov 5;45(3):735-41
references: 5001847
references: Nature. 1992 Jan 30;355(6359):472-5
references: 18481394
references: J Biol Chem. 1975 May 25;250(10):3897-903
references: 1126938
references: Org Biomol Chem. 2012 Mar 21;10(11):2248-54
references: 22307920
references: J Am Chem Soc. 2005 Aug 24;127(33):11602-3
references: 16104727
references: Appl Microbiol. 1964 Jan;12:46-52
references: 14106940
references: Structure. 1999 Jun 15;7(6):R141-6
references: 10404588
references: Curr Protein Pept Sci. 2000 Sep;1(2):209-35
references: 12369917
references: Am Rev Respir Dis. 1991 Jul;144(1):1-2
references: 2064112
references: Microbiology (Reading). 2000 Oct;146 ( Pt 10):2695-2703
references: 11021945
references: Biochemistry. 2008 Nov 4;47(44):11606-15
references: 18842002