Convergent evolution sheds light on the anti-β-elimination mechanism common to family 1 and 10 polysaccharide lyases

Charnock, Simon J.; Brown, Ian; Turkenburg, J.P.; Black, Gary W.; Davies, G.J.

doi:10.1073/pnas.182431199

Cited by 90 publications

(100 citation statements)

References 37 publications

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“…3 and 4). The argument for arginine as base is strongly supported by the recent Pel10A structure that has convergent active site geometry with the Pel1A enzymes, including an active site arginine, but a non-related architecture (35). Perhaps the most remarkable difference between the active sites of Pel9A and these other polysaccharide lyases is that in Pel9A, there is no arginine equivalent to the putative base in Pel9A (Arg-218 in PelC).…”

Section: Resultsmentioning

confidence: 99%

The Crystal Structure of Pectate Lyase Pel9A from Erwinia chrysanthemi

Jenkins

Shevchik²,

Hugouvieux‐Cotte‐Pattat³

et al. 2004

Journal of Biological Chemistry

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The "family 9 polysaccharide lyase" pectate lyase L (Pel9A) from Erwinia chrysanthemi comprises a 10-coil parallel ␤-helix domain with distinct structural features including an asparagine ladder and aromatic stack at novel positions within the superhelical structure. Pel9A has a single high affinity calcium-binding site strikingly similar to the "primary" calcium-binding site described previously for the family Pel1A pectate lyases, and there is strong evidence for a common second calcium ion that binds between enzyme and substrate in the "Michaelis" complex. Although the primary calcium ion binds substrate in subsite ؊1, it is the second calcium ion, whose binding site is formed by the coming together of enzyme and substrate, that facilitates abstraction of the C5 proton from the sacharride in subsite ؉1. The role of the second calcium is to withdraw electrons from the C6 carboxylate of the substrate, thereby acidifying the C5 proton facilitating its abstraction and resulting in an E1cb-like anti-␤-elimination mechanism. The active site geometries and mechanism of Pel1A and Pel9A are closely similar, but the catalytic base is a lysine in the Pel9A enzymes as opposed to an arginine in the Pel1A enzymes.Polysaccharide lyases (EC 4.2.2.-) are carbon-oxygen lyases that exploit ␤-elimination chemistry to cleave C5 uronic acid containing pyranoside substrates such as polygalacturonates, alginates, hyaluronan, and chondroitin. In contrast to the 90 sequence-derived families of glycoside hydrolases, polysaccharide lyases have been grouped into just 12 families (1).

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Section: Resultsmentioning

confidence: 99%

The Crystal Structure of Pectate Lyase Pel9A from Erwinia chrysanthemi

Jenkins

Shevchik²,

Hugouvieux‐Cotte‐Pattat³

et al. 2004

Journal of Biological Chemistry

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“…The guanidinium group of Arg-214 lies within hydrogen bonding distance (3.4 Å) of the N1 atom of the isoalloxazine ring of FMN and therefore could potentially act as the general acid/base necessary to protonate/ deprotonate the N1 atom during the catalytic cycle. Although uncommon, an arginine residue has been proposed to act as a catalytic base in the pectate lyase family (29) and an acid in quinol:fumarate reductase (30). However, the pH optimum of 7.4 for DCR (31) is well below that of pectate lyases, ϳpH 10.5, suggesting that Arg-214 is maintained in a protonated state in DCR.…”

Section: Discussionmentioning

confidence: 99%

The Crystal Structure and Reaction Mechanism of Escherichia coli 2,4-Dienoyl-CoA Reductase

Hubbard

Liang

Schulz

et al. 2003

Journal of Biological Chemistry

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Escherichia coli 2,4-dienoyl-CoA reductase is an ironsulfur flavoenzyme required for the metabolism of unsaturated fatty acids with double bonds at even carbon positions. The enzyme contains FMN, FAD, and a 4Fe-4S cluster and exhibits sequence homology to another ironsulfur flavoprotein, trimethylamine dehydrogenase. It also requires NADPH as an electron source, resulting in reduction of the C4-C5 double bond of the acyl chain of the CoA thioester substrate. The structure presented here of a ternary complex of E. coli 2,4-dienoyl-CoA reductase with NADP ؉ and a fatty acyl-CoA substrate reveals a possible mechanism for substrate reduction and provides details of a plausible electron transfer mechanism involving both flavins and the iron-sulfur cluster. The reaction is initiated by hydride transfer from NADPH to FAD, which in turn transfers electrons, one at a time, to FMN via the 4Fe-4S cluster. In the final stages of the reaction, the fully reduced FMN provides a hydride ion to the C5 atom of substrate, and Tyr-166 and His-252 are proposed to form a catalytic dyad that protonates the C4 atom of the substrate and complete the reaction. Inspection of the substrate binding pocket explains the relative promiscuity of the enzyme, catalyzing reduction of both 2-trans,4-cis-and 2-trans,4-trans-dienoyl-CoA thioesters.Metabolism of unsaturated fatty acids requires auxiliary enzymes in addition to those used in ␤-oxidation. After a given number of cycles through the ␤-oxidation pathway, those unsaturated fatty acyl-CoAs with double bonds at even-numbered carbon positions contain 2-trans,4-cis double bonds that cannot be modified by enoyl-CoA hydratase. Therefore, an auxiliary enzyme, 2,4-dienoyl-CoA reductase (DCR 1 ; EC 1.3.1.34), is used that utilizes NADPH to remove the C4-C5 double bond (1, 2). DCR is unusual in that it lacks stereospecificity, catalyzing the reduction of both natural fatty acids with cis double bonds, as well as substrates containing trans double bonds (3).Two structurally unrelated forms of DCR have been isolated, both of which use NADPH as reducing equivalents to catalyze reduction of a 2,4-dienoyl-CoA to an enoyl-CoA. The Escherichia coli enzyme contains both FMN and FAD noncovalently bound to a single polypeptide, reducing the substrate by a hydride transfer mechanism in which reducing equivalents from NADPH are supplied indirectly to substrate (1). It functions as a monomer, having a molecular mass of 73 kDa, and has been shown recently to contain a 4Fe-4S cluster (4). The enzyme produces 2-trans-enoyl-CoA (1), which can be incorporated into the ␤-oxidation pathway. In contrast, three homologous eukaryotic forms have been identified, two mitochondrial and one peroxisomal. Two of these lack flavin and iron-sulfur cofactors but are homotetramers with a total molecular mass of 124 kDa. (The third has not been characterized.) The substrate is reduced by a direct hydride transfer mechanism from NADPH to form trans-3-enoyl-CoA (1). Thus, eukaryotic DCR requires another auxiliary enzyme, ⌬ 3 ,⌬ 2 -enoy...

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“…In general, these enzymes display a right-handed parallel b-helix topology. Exceptions include PL10 pectate lyases, which adopt an (a/a) 6 toroid conformation (Charnock et al, 2002b), and PL2 lyases, which display a (a/a) 7 barrel and utilize manganese rather than calcium in the active site (Abbott and Boraston, 2007). The catalytic apparatus in PL10, and those displaying a b-helix fold, is conserved, providing an example of convergent evolution (Charnock et al, 2002b).…”

Section: Pectin Degradationmentioning

confidence: 99%