Molecular Recognition and Interfacial Catalysis by the Essential Phosphatidylinositol Mannosyltransferase PimA from Mycobacteria

Guerin, Marcelo E.; Korduláková, Jana; Schaeffer, Francis; Svetlíková, Zuzana; Buschiazzo, Alejandro; Giganti, David; Gicquel, Brigitte; Mikušová, Katarı́na; Jackson, Mary; Alzari, Pedro M.

doi:10.1074/jbc.m702087200

Cited by 123 publications

(232 citation statements)

References 51 publications

(50 reference statements)

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“…of 2.10 Å, and 21% sequence identity) and the phospatidylinositol mannosyltransferase PimA (Z-score of 9.4, r.m.s.d. of 2.14 Å, 26% sequence identity) (28,29). A slightly lower score was observed for the only other GT-4 family member with a known structure, an enzyme involved in avilamycin A biosynthesis, AviGT4 (Z-score of 7.4, r.m.s.d.…”

Section: Resultsmentioning

confidence: 99%

“…The structures of WaaG and OtsA were determined with UDP-2-deoxy-2-fluoroglucose (UDP-2FGlc) (28,44), and PimA was determined with GDPmannose (GDP-man) (29). The binding mode of UDP-2FGlc to Electron density for 1-L-Ins-1-P was sufficient only to fit in one subunit but was modeled in the dimer shown here for illustrative purposes.…”

Section: Resultsmentioning

confidence: 99%

“…Molecular replacement searches for the missing C-terminal domain after placement of the N-terminal domains were unsuccessful. The recently determined structure of PimA (PDB ID: 2GEJ) was used as a guide for the structure of a closed GT-B:GT-4 glycosyltransferase family member (29). Utilizing electron density maps phased with only the two placed N-terminal domains, and the PimA structure as a guide, there was enough secondary structure visible in a skeletonized map to manually place both C-terminal domains, which were then successfully refined into their correct positions with rigid body refinement.…”

Section: Methodsmentioning

confidence: 99%

“…GT4 is the second largest CAZy data base family (6563 CAZY entries at the time of this writing) and have a diverse set of donor and acceptor molecules. Only recently a limited number of GT4 family members have been structural characterized, including WaaG, which transfers glucose from UDP-glucose onto L-glycero-D-mannoheptose II; AviGT4, which is involved in avilamycin A biosynthesis; and PimA, a phosphatidylinositol mannosyltransferase (28,29).…”

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confidence: 99%

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Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

Vetting

Frantom

Blanchard

2008

Journal of Biological Chemistry

110

192

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The glycosyltransferase termed MshA catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to 1-Lmyo-inositol-1-phosphate in the first committed step of mycothiol biosynthesis. The structure of MshA from Corynebacterium glutamicum was determined both in the absence of substrates and in a complex with UDP and 1-L-myo-inositol-1-phosphate. MshA belongs to the GT-B structural family whose members have a two-domain structure with both domains exhibiting a Rossman-type fold. Binding of the donor sugar to the C-terminal domain produces a 97°rotational reorientation of the N-terminal domain relative to the C-terminal domain, clamping down on UDP and generating the binding site for 1-Lmyo-inositol-1-phosphate. The structure highlights the residues important in binding of UDP-N-acetylglucosamine and 1-L-myo-inositol-1-phosphate. Molecular models of the ternary complex suggest a mechanism in which the ␤-phosphate of the substrate, UDP-N-acetylglucosamine, promotes the nucleophilic attack of the 3-hydroxyl group of 1-L-myo-inositol-1-phosphate while at the same time promoting the cleavage of the sugar nucleotide bond.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

Vetting

Frantom

Blanchard

2008

Journal of Biological Chemistry

110

192

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“…1S). As had been the case with the M. tuberculosis PimA protein earlier (5,14,17), attempts to produce the PimBЈ enzyme from M. tuberculosis yielded relatively small amounts of soluble protein compared with the M. smegmatis version, and these efforts were thus not pursued further.…”

Section: Mspimbј Catalyzes In Vitro the Transfer Of A Manp Residue Tomentioning

confidence: 99%

New Insights into the Early Steps of Phosphatidylinositol Mannoside Biosynthesis in Mycobacteria

Guerin

Kaur

Somashekar

et al. 2009

Journal of Biological Chemistry

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103

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Phosphatidyl-myo-inositol mannosides (PIMs) are key glycolipids of the mycobacterial cell envelope. They are considered not only essential structural components of the cell but also important molecules implicated in host-pathogen interactions. Although their chemical structures are well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still incomplete. Here we show for the first time that although both mannosyltransferases PimA and PimB (MSMEG_4253) recognize phosphatidyl-myo-inositol (PI) as a lipid acceptor, PimA specifically catalyzes the transfer of a Manp residue to the 2-position of the myo-inositol ring of PI, whereas PimB exclusively transfers to the 6-position. Moreover, whereas PimB can catalyze the transfer of a Manp residue onto the PI-monomannoside (PIM 1 ) product of PimA, PimA is unable in vitro to transfer Manp onto the PIM 1 product of PimB. Further assays using membranes from Mycobacterium smegmatis and purified PimA and PimB indicated that the acylation of the Manp residue transferred by PimA preferentially occurs after the second Manp residue has been added by PimB. Importantly, genetic evidence is provided that pimB is an essential gene of M. smegmatis. Altogether, our results support a model wherein Ac 1 PIM 2 , a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA, followed by PimB, and finally the acyltransferase MSMEG_2934. The essentiality of these three enzymes emphasizes the interest of novel anti-tuberculosis drugs targeting the initial steps of PIM biosynthesis. PIMs3 are unique mannolipids found in abundant quantities in the inner and outer membranes of the cell envelope of Mycobacterium spp. and a few other actinomycetes. 4 They are based on a phosphatidyl-myo-inositol (PI) lipid anchor carrying one to six Manp residues and up to four acyl chains (for review see Refs. 1, 2). Based on a conserved mannosyl-PI anchor, they are also thought to be the precursors of the two major mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) (1, 2). PIMs, LM, and LAM are considered not only essential structural components of the mycobacterial cell envelope (3-6), but also important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy (1).Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still fragmentary. According to the currently accepted model, the biosynthetic pathway is initiated by the transfer of two Manp residues and a fatty acyl chain to PI in the cytoplasmic leaflet of the plasma membrane. Based on genetic and biochemical evidence, Korduláková et al. (5) identified PimA (MSMEG_2935 in Mycobacterium smegmatis mc 2 155) as the enzyme that catalyzes the first mannosylation step of the pathway transferring a Manp residue most likely to the 2-position of the myo-inositol (myo-Ins) ring of PI. In contrast, the identity of PimBЈ, the enzyme responsible for the tran...

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Carbohydrate synthesis by disaccharide phosphorylases: Reactions, catalytic mechanisms and application in the glycosciences

Luley‐Goedl

Nidetzky

2010

Biotechnology Journal

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Disaccharide phosphorylases are glycosyltransferases (EC 2.4.1.α) of specialized carbohydrate metabolism in microorganisms. They catalyze glycosyl transfer to phosphate using a disaccharide as donor substrate. Phosphorylases for the conversion of naturally abundant disaccharides including sucrose, maltose, α,α‐trehalose, cellobiose, chitobiose, and laminaribiose have been described. Structurally, these disaccharide phosphorylases are often closely related to glycoside hydrolases and transglycosidases. Mechanistically, they are categorized according the stereochemical course of the reaction catalyzed, whereby the anomeric configuration of the disaccharide donor substrate may be retained or inverted in the sugar 1‐phosphate product. Glycosyl transfer with inversion is thought to occur through a single displacement‐like catalytic mechanism, exemplified by the reaction coordinate of cellobiose/chitobiose phosphorylase. Reaction via configurational retention takes place through the double displacement‐like mechanism employed by sucrose phosphorylase. Retaining α,α‐trehalose phosphorylase (from fungi) utilizes a different catalytic strategy, perhaps best described by a direct displacement mechanism, to achieve stereochemical control in an overall retentive transformation. Disaccharide phosphorylases have recently attracted renewed interest as catalysts for synthesis of glycosides to be applied as food additives and cosmetic ingredients. Relevant examples are lacto‐N‐biose and glucosylglycerol whose enzymatic production was achieved on multikilogram scale. Protein engineering of phosphorylases is currently pursued in different laboratories with the aim of broadening the donor and acceptor substrate specificities of naturally existing enzyme forms, to eventually generate a toolbox of new catalysts for glycoside synthesis.

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Molecular Recognition and Interfacial Catalysis by the Essential Phosphatidylinositol Mannosyltransferase PimA from Mycobacteria

Cited by 123 publications

References 51 publications

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

New Insights into the Early Steps of Phosphatidylinositol Mannoside Biosynthesis in Mycobacteria

Carbohydrate synthesis by disaccharide phosphorylases: Reactions, catalytic mechanisms and application in the glycosciences

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