Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity.

Karina Verdel‐Aranda,Susana T López‐Cortina,Francisco Barona‐Gómez,David A Hodgson

doi:10.1111/1751-7915.12175

Abstract

The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications.

Highlights

It is well acknowledged that enzymes can be promiscuous or multifunctional, catalysing different chemical transformations upon one or more substrates, or catalysing identical reactions using several related or unrelated substrates (O’Brien and Herschlag, 1999; Khersonsky et al, 2006; Khersonsky and Tawfik, 2010)
Since the recognition of this phenomenon, enzyme promiscuity has been defined as the ability of enzymes to exert other activities beyond those for which they have evolved, implying that such activities are overall not relevant for the physiology of the organism (Copley, 2003; Khersonsky et al, 2006; Khersonsky and Tawfik, 2010)
Enzyme promiscuity followed by enzyme recruitment, seems to have given rise to many peripheral metabolic pathways, such as natural products biosynthetic pathways (Vining, 1992)

Summary

Introduction

It is well acknowledged that enzymes can be promiscuous or multifunctional, catalysing different chemical transformations upon one or more substrates, or catalysing identical reactions using several related or unrelated substrates (O’Brien and Herschlag, 1999; Khersonsky et al, 2006; Khersonsky and Tawfik, 2010). It could be argued that one of the main challenges in molecular biology is the correct functional annotation of proteins, a situation that is accentuated in the context of promiscuous enzymes. This challenge has been addressed either by reductionist studies focusing in a single protein, or after high-throughput systems-level analyses involving many proteins (Laskowski et al, 2005; Redfern et al, 2009; Schnoes et al, 2013). The trade-off between these approaches represents a conundrum for annotation of enzyme superfamilies, defined as structurally and functionally related enzymes that can catalyse similar reactions upon quite different substrates (Gerlt and Babbitt, 2001; Gerlt et al, 2012). A deep understanding of the relationships among sequence, structure and function of enzyme superfamilies is limited to few cases, such as the enolase superfamily (Gerlt et al, 2012)

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