Crystal structure of <i>Bacillus subtilis</i> ioli shows endonuclase IV fold with altered Zn binding

Zhang, R.-G.; Dementieva, I.; Duke, N.E.C.; Collart, F.; Quaite-Randall, Elsie; Alkire, R. W.; Dieckman, Lynda; Maltsev, Natalia; Korolev, O.; Joachimiak, Andrzej

doi:10.1002/prot.10159

Cited by 17 publications

(18 citation statements)

References 12 publications

(6 reference statements)

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“…iolI is the ninth gene of the B. subtilis iol operon, encoding a conserved 278-residue protein of unknown function. Its crystal structure is reported to have a beta-barrel (TIM) configuration, suggestive of structural homology to both endonuclease IV and xylose isomerase with a Zn 2ϩ -binding fold (24). In this study, we found that iolI encoded an inosose isomerase functioning for the reversible interconversion of 2KMI and 1KDCI (Fig.…”

Section: Resultsmentioning

confidence: 59%

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Genetic Modification of Bacillus subtilis for Production of d - chiro -Inositol, an Investigational Drug Candidate for Treatment of Type 2 Diabetes and Polycystic Ovary Syndrome

Yoshida

Yamaguchi

Morinaga

et al. 2006

Appl Environ Microbiol

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D-chiro-Inositol (DCI) is a drug candidate for the treatment of type 2 diabetes and polycystic ovary syndrome, since it improves the efficiency with which the body uses insulin and also promotes ovulation. Here, we report genetic modification of Bacillus subtilis for production of DCI from myo-inositol (MI). The B. subtilis iolABC-DEFGHIJ operon encodes enzymes for the multiple steps of the MI catabolic pathway. In the first and second steps, MI is converted to 2-keto-MI (2KMI) by IolG and then to 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione by IolE. In this study, we identified iolI encoding inosose isomerase, which converts 2KMI to 1-keto-D-chiroinositol (1KDCI), and found that IolG reduces 1KDCI to DCI. Inactivation of iolE in a mutant constitutively expressing the iol operon blocked the MI catabolic pathway to accumulate 2KMI, which was converted to DCI via the activity of IolI and IolG. The mutant was able to convert at least 6% of input MI in the culture medium to DCI.

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Section: Resultsmentioning

confidence: 59%

“…2A), but it did not react with 1KLCI. The enzyme required metal ions such as Mn 2ϩ , Fe 2ϩ , or Co 2ϩ for its activity, with a preference for Mn 2ϩ , despite previous structural analysis demonstrating its capacity to bind Zn 2ϩ (24). The enzyme showed a pH optimum of between pH 7.5 to 8.5.…”

Section: Resultsmentioning

confidence: 84%

Genetic Modification of Bacillus subtilis for Production of d - chiro -Inositol, an Investigational Drug Candidate for Treatment of Type 2 Diabetes and Polycystic Ovary Syndrome

Yoshida

Yamaguchi

Morinaga

et al. 2006

Appl Environ Microbiol

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“…AsbF belongs to AP endonuclease 2 TIM barrel proteins (19), with the closest homologues including xylose isomerase and the myoinositol catabolism protein IolI (20). The internal section of AsbF is well packed by relatively large side chains with a narrow bottom part of the barrel relative to the top.…”

Section: Structural Characterization Of Asbf Bound To Its Product 3mentioning

confidence: 99%

Structural and functional analysis of AsbF: Origin of the stealth 3,4-dihydroxybenzoic acid subunit for petrobactin biosynthesis

Pfleger

Kim

Nusca

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Petrobactin, a virulence-associated siderophore produced by Bacillus anthracis, chelates ferric iron through the rare 3,4-isomer of dihydroxybenzoic acid (3,4-DHBA). Most catechol siderophores, including bacillibactin and enterobactin, use 2,3-DHBA as a biosynthetic subunit. Significantly, siderocalin, a factor involved in human innate immunity, sequesters ferric siderophores bearing the more typical 2,3-DHBA moiety, thereby impeding uptake of iron by the pathogenic bacterial cell. In contrast, the unusual 3,4-DHBA component of petrobactin renders the siderocalin system incapable of obstructing bacterial iron uptake. Although recent genetic and biochemical studies have revealed selected early steps in petrobactin biosynthesis, the origin of 3,4-DHBA as well as the function of the protein encoded by the final gene in the B. anthracis siderophore biosynthetic (asb) operon, asbF (BA1986), has remained unclear. In this study we demonstrate that 3,4-DHBA is produced through conversion of the common bacterial metabolite 3-dehydroshikimate (3-DHS) by AsbF-a 3-DHS dehydratase. Elucidation of the cocrystal structure of AsbF with 3,4-DHBA, in conjunction with a series of biochemical studies, supports a mechanism in which an enolate intermediate is formed through the action of this 3-DHS dehydratase metalloenzyme. Structural and functional parallels are evident between AsbF and other enzymes within the xylose isomerase TIM-barrel family. Overall, these data indicate that microbial species shown to possess homologs of AsbF may, like B. anthracis, also rely on production of the unique 3,4-DHBA metabolite to achieve full viability in the environment or virulence within the host.AsbF structure ͉ dehydratase ͉ siderophore ͉ virulence factor S iderophore production in pathogenic bacteria has gained considerable attention because of its crucial function in essential iron uptake by many microbes and the relevance of siderophore-associated proteins as molecular markers of various infectious agents (1). In Bacillus anthracis, the causative agent of anthrax, 2 siderophores, petrobactin and bacillibactin (Fig. 1A), play a significant role during iron acquisition (2-4), but only petrobactin is absolutely essential for full virulence within a mammalian host (5). This siderophore was initially discovered from the Gram-negative marine bacterium Marinobacter hydrocarbonoclasticus, whose genome bears a biosynthetic gene cluster homologous to the B. anthracis asb operon (2). Recent genetic and chemical analysis suggests that petrobactin biosynthesis may also be a prerequisite for virulence in related Bacillus species (6). These studies highlight the importance of elucidating the mechanisms of siderophore production in pathogenic microbes as a target for abrogating infection by organisms like B. anthracis, a rapidly virulent microbe with proven potential as a bioterrorism agent. Based on these factors, we have initiated studies to investigate key biosynthetic enzymes for petrobactin assembly in efforts to establish new antimicrobial targets t...

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“…Similar coordination of the substrate oxygen was observed in D-psicose 3-epimerase (18) and in L-rhamnose isomerase (42). There are also similar metalbinding sites in the structural homologs IolI (PDB code 1I6N) (43) and endonuclease IV (16).…”

Section: ϫmentioning

confidence: 61%

Structure of l -Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic l -Ascorbate Utilization Pathway of Escherichia coli : Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

et al. 2008

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Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of L-ascorbate under anaerobic conditions. UlaD catalyzes a ␤-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel ␤-strands. The enzyme binds Zn 2؉ , which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the ␤1/␣1 loop and ␣3 helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands ␤7 and ␤8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.It has been known for nearly 70 years that various bacteria, including Escherichia coli, can ferment L-ascorbate (vitamin C) under anaerobic conditions (2,10,30,40). This process has been demonstrated to involve proteins encoded by two divergently transcribed operons, the ulaGR and ulaABCDEF operons (3,40,44). The ulaG gene encodes L-ascorbate-6-phosphate lactonase. The ulaABCDEF operon encodes an L-ascorbate phosphotransferase transport system (UlaABC, formerly designated SgaTBA) responsible for the uptake and phosphorylation of L-ascorbate, and the ulaDEF genes encode three enzymes that catalyze the conversion of the phosphorylated, hydrolytic product of L-ascorbate, 3-keto-L-gulonate-6-phosphate, to D-xylulose-5-phosphate, which is subsequently metabolized by the pentose phosphate pathway. UlaD (formerly SgaH) decarboxylates 3-keto-L-gulonate-6-phosphate, yielding L-xylulose-5-phosphate, which is subsequently epimerized by UlaE (formerly SgaU) at C-3 to L-ribulose-5-phosphate and then epimerized by UlaF (formerly SgaE) at C-4 to D-xylulose-5-phosphate (Fig.

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Crystal structure of Bacillus subtilis ioli shows endonuclase IV fold with altered Zn binding

Cited by 17 publications

References 12 publications

Genetic Modification of Bacillus subtilis for Production of d - chiro -Inositol, an Investigational Drug Candidate for Treatment of Type 2 Diabetes and Polycystic Ovary Syndrome

Genetic Modification of Bacillus subtilis for Production of d - chiro -Inositol, an Investigational Drug Candidate for Treatment of Type 2 Diabetes and Polycystic Ovary Syndrome

Structural and functional analysis of AsbF: Origin of the stealth 3,4-dihydroxybenzoic acid subunit for petrobactin biosynthesis

Structure of l -Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic l -Ascorbate Utilization Pathway of Escherichia coli : Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

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