Characterization of the N-acetyl-α-D-glucosaminyl l-malate synthase and deacetylase functions for bacillithiol biosynthesis in Bacillus anthracis .
Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce α-d-glucosaminyl l-malate (GlcN-malate) from UDP-GlcNAc and l-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (→GlcNAc-malate) and the BaBshB deacetylase (→GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of l-malate, but not other α-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-d-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.
Parsonage
Derek
D
Center for Structural Biology, Wake Forest University School of Medicine,Winston-Salem, North Carolina 27157, USA.
Newton
Gerald L
GL
Holder
Robert C
RC
Wallace
Bret D
BD
Paige
Carleitta
C
Hamilton
Chris J
CJ
Dos Santos
Patricia C
PC
Redinbo
Matthew R
MR
Reid
Sean D
SD
Claiborne
Al
A
D001408:Bacillus anthracis / Q000201:enzymology* / Q000378:metabolism; D001665:Binding Sites; D001894:Borohydrides; D003545:Cysteine / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D005944:Glucosamine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D006020:Glycopeptides; D016695:Glycosyltransferases / Q000096:biosynthesis / Q000378:metabolism; D007294:Inositol; D019753:Intramolecular Lyases; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014530:Uridine Diphosphate / Q000096:biosynthesis / Q000378:metabolism
D001894:Borohydrides; D006020:Glycopeptides; D013438:Sulfhydryl Compounds; C543521:bacillithiol; C089265:mycothiol; C014651:mercaptoundecahydrododecaborate; D007294:Inositol; D014530:Uridine Diphosphate; D016695:Glycosyltransferases; D019753:Intramolecular Lyases; C105497:D-myo-inositol-3-phosphate synthase; D003545:Cysteine; D005944:Glucosamine
J Bacteriol. 2005 Dec;187(23):8055-62
16291679
Glycobiology. 2003 Oct;13(10):707-12
12881407
FEMS Microbiol Lett. 1990 Jun 1;57(3):245-8
2170230
Genes Genet Syst. 2009 Aug;84(4):253-67
20057163
Protein Expr Purif. 2006 Jun;47(2):542-50
16630724
J Bacteriol. 2006 Sep;188(17):6092-100
16923876
Biochemistry. 2003 Oct 21;42(41):12067-76
14556638
J Bacteriol. 2009 Dec;191(24):7520-30
19837798
Appl Environ Microbiol. 2007 Feb;73(3):846-54
17122389
Structure. 1999 Mar 15;7(3):309-17
10368300
Methods Mol Biol. 1995;47:185-93
7550734
Bioinformatics. 2000 Jun;16(6):566-7
10980157
FEBS J. 2007 Jun;274(12):3044-54
17501983
Antioxid Redox Signal. 2003 Aug;5(4):403-11
13678528
Biochemistry. 1996 Jun 11;35(23):7341-55
8652510
Science. 2001 Jan 26;291(5504):643-6
11158675
Biochemistry. 2002 Jun 4;41(22):6885-90
12033919
J Bacteriol. 2007 Jun;189(11):3996-4013
17384197
Biochemistry. 2007 Mar 20;46(11):3234-45
17323930
Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):9109-14
19451637
J Biol Chem. 2003 Nov 21;278(47):47166-70
12958317
J Biol Chem. 1998 Mar 6;273(10):5744-51
9488707
Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6482-6
20308541
Angew Chem Int Ed Engl. 2010 Feb 8;49(7):1234-7
20077550
Methods Enzymol. 1997;276:307-26
27754618
Infect Immun. 2006 Mar;74(3):1949-53
16495572
Biochem J. 1999 Apr 1;339 ( Pt 1):185-92
10085243
Bioinformatics. 1999 Apr;15(4):305-8
10320398
Nucleic Acids Res. 2009 Jan;37(Database issue):D233-8
18838391
Microbiol Mol Biol Rev. 2008 Sep;72(3):471-94
18772286
J Biol Chem. 2008 Jun 6;283(23):15834-44
18390549
Appl Microbiol Biotechnol. 2007 Feb;74(1):43-9
17021870
J Biol Chem. 2007 Jul 13;282(28):20705-14
17510062
J Bacteriol. 2008 Sep;190(18):6271-5
18641144
Mol Microbiol. 2003 Mar;47(6):1723-32
12622824
Biochemistry. 1999 Mar 2;38(9):2725-37
10052943
J Biol Chem. 2010 Jan 15;285(3):1587-96
19917605
J Chem Theory Comput. 2009 Dec 8;5(12):3299-311
26602510
J Appl Bacteriol. 1974 Jun;37(2):265-7
4212901
Nat Chem Biol. 2009 Sep;5(9):625-7
19578333
Anal Biochem. 1994 Jul;220(1):92-7
7978262
Annu Rev Microbiol. 2009;63:575-97
19575568
J Bacteriol. 1983 Jan;153(1):475-84
6401287
EMBO J. 2002 Jun 3;21(11):2778-87
12032090
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
FEBS J. 2010 Jul;277(13):2740-53
20491912
J Bacteriol. 2009 May;191(10):3203-11
19304856
Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
20124702
Biochemistry. 2009 Mar 10;48(9):1847-9
19196010
J Agric Food Chem. 2005 May 18;53(10):4149-56
15884853
J Biol Chem. 1997 Jun 20;272(25):15661-7
9188456
J Biol Chem. 2006 Nov 10;281(45):33910-20
16940050
Proteins. 2008 Nov 15;73(3):784-7
18712829
Proc Natl Acad Sci U S A. 2008 May 6;105(18):6735-40
18456836
Biochemistry. 1966 May;5(5):1690-5
4289778
Biochemistry. 2006 Sep 26;45(38):11278-89
16981688
J Bacteriol. 2007 Apr;189(8):3296-301
17277068
J Bacteriol. 2002 Aug;184(16):4555-72
12142426
J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
19461840
J Bacteriol. 2001 Apr;183(7):2380-3
11244082
J Biol Chem. 1998 Mar 6;273(10):5752-7
9488708
FEBS J. 2007 Jun;274(11):2778-87
17459099
Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
7984417
J Biol Chem. 1998 Nov 13;273(46):30391-7
9804803
Biochemistry. 2009 Oct 13;48(40):9650-67
19725515
Nat Prod Rep. 2008 Dec;25(6):1091-117
19030604
Protein Expr Purif. 2005 May;41(1):207-34
15915565
Infect Immun. 2007 Jul;75(7):3434-44
17470545
Biochem Soc Trans. 2003 Jun;31(Pt 3):563-71
12773157
Biochemistry. 2008 May 6;47(18):5182-93
18399646