Enzyme Mechanism – A Structural Perspective 563 Structural insights into the evolution of the pantothenate-biosynthesis pathway C.M.C. Lobley* 1 , F. Schmitzberger*, M.L. Kilkenny*, H. Whitney†, H.H. Ottenhof†, E. Chakauya†, M.E. Webb‡, L.M. Birch‡, K.L. Tuck‡, C. Abell‡, A.G. Smith† and T.L. Blundell* *Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, U.K., †Department of Plant Sciences, Downing Street, Cambridge CB2 3EA, U.K., and ‡University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K. Abstract Pantothenate is synthesized in bacteria, fungi and plants, and as vitamin B5 is a dietary requirement in animals. The three-dimensional structures of the four Escherichia coli enzymes involved in the production of pantothenate have been determined. We describe the use of comparative analyses of the sequences and structures to identify distant homologues of the four enzymes in an attempt to understand the evolution of the pathway. We conclude that it is likely to have evolved via a patchwork mechanism, whereby the individual enzymes were recruited separately. Introduction Pantothenate is a necessary precursor to coenzyme A and phosphopantetheine, the prosthetic group of the acyl carrier protein, both of which are vital to a multitude of metabolic processes [1]. Also known as vitamin B5, it is a dietary requirement in animals. However, pantothenate is synthesized by bacteria, fungi and plants. The enzymes that are required to produce pantothenate in Escherichia coli have been extensively studied by X-ray crystallography ([3–5], and F. von Delft, T. Inoue, A.S. Saldanha, H.H. Ottenhof, F. Schmitzberger, L.M. Birch, V. Dhanaraj, M. Witty, C. Abell, A.G. Smith and T.L. Blundell, unpublished work) and kinetic analysis [6–8], and are attractive anti-microbial, fungicide and herbicide targets. In E. coli, four enzymes are involved in the production of pantothenate (Scheme 1). Ketopantoate hydroxymethyl- transferase (KPHMT; EC 2.1.2.11, SwissProt P31057) converts α-ketoisovalerate into ketopantoate using 5,10- methylene tetrahydrofolate. Subsequently ketopantoate is reduced to pantoate by ketopantoate reductase (KPR; EC 1.1.1.169, SwissProt P77728) using NADPH as the hydrogen donor. Concomitantly L-aspartate is converted into β -alanine by aspartate decarboxylase (ADC; EC 4.1.1.11, SwissProt P31664). The ATP-consuming condensation of β -alanine and pantoate catalysed by pantothenate synthetase (PS; EC 6.3.2.1, SwissProt P31663) completes the pathway. This simple, well-studied pathway is ideal for the investigation of evolution not only of the enzymes individually but also of the pathway as a whole. Key words: enzyme pathway, evolution, pantothenate, X-ray crystallography. Abbreviations used: KPHMT, ketopantoate hydroxymethyltransferase; KPR, ketopantoate reductase; ADC, aspartate decarboxylase; PS, pantothenate synthetase; PEPM, phosphoenol- pyruvate mutase; ICL, isocitrate lyase; CENDH, N-(1-d-carboxyethyl)-l-norvaline dehydrogenase; AHIR, acetohydroxy isomeroreductase; G3PCT, glucose-3-phosphate cytidylyl transferase. 1 To whom correspondence should be addressed (e-mail carina@cryst.bioc.cam.ac.uk). To date, all theories of enzyme-pathway evolution have assumed that the first organisms could survive solely on the prebiotic soup – the heterotrophic origin of life. The possible chemical compositions of the prebiotic soup and early atmosphere are the subject of some debate, but the potential availability of pantothenate and its precursors has been shown experimentally [9]. The Horowitz hypothesis (retrograde evolution) assumes that when a metabolite from the prebiotic soup is exhausted, existing enzymes mutate to give a new enzyme that can catalyse the synthesis of the necessary metabolite. When that intermediate becomes limiting the first enzyme will again mutate and a pathway will begin to emerge to produce the required metabolite. If this were the case, each enzyme in a pathway would be homologous [10]. This appears to have happened in the tryptophan-biosynthesis pathway, where three sequential enzymes have similar structures and active sites. All three proteins are members of the same superfamily in the SCOP (Structural Classification of Proteins) database [11], implying a common ancestor. The patchwork hypothesis, proposed by Ycas in 1974 [12] and adapted by Jenson in 1976 [13], relies on the existence of a pool of enzymes with low specificity. These enzymes were then recruited to a pathway as required, and the specificity was honed to give the highly specific enzymes we see today. This cannot apply to the very first pathways, since it requires the emergence of protein biosynthesis [12,13]. These classical views of evolving pathways were comple- mented by theoretical models of gene duplication, pioneered by Susumo Ohno [14], in which functionally redundant paralogous genes were assumed to be produced by whole- genome duplication, non-homologous recombination or through transpositions, thereby freeing one from selective constraints and allowing rapid evolution, initially by neutral evolution. Such redundancy will normally be eliminated by mutations causing one copy to become non-functional, but C  2003 Biochemical Society