Reversing Evolution: Re-establishing Obligate Metal Ion Dependence in a Metal-independent KDO8P Synthase

Cochrane, Fiona C.; Cookson, T.V.M.; Jameson, Geoffrey B.; Parker, Emily J.

doi:10.1016/j.jmb.2009.05.014

Cited by 15 publications

(39 citation statements)

References 40 publications

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“…In the HMM vector of Figure 1A, position numbers refer to the reference sequence of Nm. KDO8PS [6]. There is a “wavy” pattern in the histogram derived from the MSA of all KDO8PSs (1 st inset of Figure 1A), which is clearly evident in the superimposed Loess [27], [28] fit.…”

Section: Resultsmentioning

confidence: 90%

“…The generality of this stability-function tradeoff must be viewed within the context of the fact that regardless of their effect on functions most mutations are destabilizing [21], [22], [23], [24]. With respect to KDO8PS, several attempts were made (e.g., [6], [11]) to map the evolutionary paths from metallo- to non-metallo forms (and vice versa) without giving sufficient consideration to the fact that sequence differences between and within these forms in different bacterial backgrounds, may be related not only to activity but also to stability.…”

Section: Resultsmentioning

confidence: 99%

“…This family contains the ‘C23’ metallo sub-family (175 sequences with Cys at position 23 of Neisseria meningitidis ( Nm. ) KDO8PS (Uniref Q9JZ55, PDB 2QKF), used here as reference [6]), and the ‘N23’ non-metallo sub-family (173 sequences with Asn at position 23). Only the positions of the MSA corresponding to the sequence of Nm.…”

Section: Resultsmentioning

confidence: 99%

“…The enzyme exists in two forms, differing in the requirement (or lack thereof) of a divalent metal for activity [2]. We and others have determined high-resolution structures of both metallo- and non-metallo forms of KDO8PS [2], [3], [4], [5], [6], which revealed the features of the active site: in metal dependent KDO8PSs the divalent metal ion (Zn 2+ , or Fe 2+ , [7]) is coordinated by the side chains of a cysteine, a histidine, a glutamic acid, and an aspartic acid. In non-metallo KDO8PSs an asparagine replaces the metal binding cysteine and there is no metal ion.…”

Section: Introductionmentioning

confidence: 99%

“…In non-metallo KDO8PSs an asparagine replaces the metal binding cysteine and there is no metal ion. Several mutagenesis studies [6], [8], [9], [10], [11], [12] and more recently, quantum mechanical simulations [7], [13], [14], have established that substitution of the metal binding cysteine with asparagine is absolutely required to convert metal-dependent to metal-independent KDO8PSs, although additional changes in the highly conserved CysAspGlyPro motif of the loop that contains the metal binding aspartic acid are necessary to achieve appreciable levels of activity [6], [12]. These observations are intriguing and raise the possibility that during the evolution of KDO8PS, after the initial choice between cysteine + metal and asparagine, additional sequence divergence occurred primarily to maximize the stability of the protein in the environment of hundreds of different bacteria.…”

Section: Introductionmentioning

confidence: 99%

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The Contribution of Coevolving Residues to the Stability of KDO8P Synthase

Ackerman

Gatti

2011

PLoS ONE

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BackgroundThe evolutionary tree of 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes a key step in the biosynthesis of bacterial endotoxin, is evenly divided between metal and non-metal forms, both having similar structures, but diverging in various degrees in amino acid sequence. Mutagenesis, crystallographic and computational studies have established that only a few residues determine whether or not KDO8PS requires a metal for function. The remaining divergence in the amino acid sequence of KDO8PSs is apparently unrelated to the underlying catalytic mechanism.Methodology/Principal FindingsThe multiple alignment of all known KDO8PS sequences reveals that several residue pairs coevolved, an indication of their possible linkage to a structural constraint. In this study we investigated by computational means the contribution of coevolving residues to the stability of KDO8PS. We found that about 1/4 of all strongly coevolving pairs probably originated from cycles of mutation (decreasing stability) and suppression (restoring it), while the remaining pairs are best explained by a succession of neutral or nearly neutral covarions.Conclusions/SignificanceBoth sequence conservation and coevolution are involved in the preservation of the core structure of KDO8PS, but the contribution of coevolving residues is, in proportion, smaller. This is because small stability gains or losses associated with selection of certain residues in some regions of the stability landscape of KDO8PS are easily offset by a large number of possible changes in other regions. While this effect increases the tolerance of KDO8PS to deleterious mutations, it also decreases the probability that specific pairs of residues could have a strong contribution to the thermodynamic stability of the protein.

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Section: Resultsmentioning

confidence: 90%