Two (βα)₈-Barrel Enzymes of Histidine and Tryptophan Biosynthesis Have Similar Reaction Mechanisms and Common Strategies for Protecting Their Labile Substrates^,

Abstract

The enzymes N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase (HisA) and phosphoribosylanthranilate isomerase (TrpF) are sugar isomerases that are involved in histidine and tryptophan biosynthesis, respectively. Both enzymes have the (betaalpha)(8)-barrel fold and catalyze Amadori rearrangements of a thermolabile aminoaldose into the corresponding aminoketose. To identify those amino acids that are essential for catalysis, conserved residues at the active sites of both HisA and TrpF from the hyperthermophile Thermotoga maritima were replaced by site-directed mutagenesis, and the purified variants were investigated by steady-state enzyme kinetics. Aspartate 8, aspartate 127, and threonine 164 appeared to be important for the HisA reaction, whereas cysteine 7 and aspartate 126 appeared to be important for the TrpF reaction. On the basis of these results and the X-ray structure of a complex between TrpF and a bound product analogue, a reaction mechanism involving general acid-base catalysis and a Schiff base intermediate is proposed for both enzymes. A comparison of the HisA and TrpF enzymes from T. maritima and Escherichia coli showed that, at the physiological temperatures of 80 and 37 degrees C, respectively, the enzymes from the hyperthermophile have significantly higher catalytic efficiencies than the corresponding enzymes from mesophiles. These results suggest that HisA and TrpF have similar chemical reaction mechanisms and use the same strategy to prevent the loss of their thermolabile substrates.

Establishing wild-type levels of catalytic activity on natural and artificial (βα) 8 -barrel protein scaffolds

“…However, neither of the 2 variants converted ProFAR into NЈ-[(5Ј-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) at a detectable rate. This result was not unexpected, because aspartate 127, which is replaced by valine in both HisAFcomIII and HisA-II, is essential for the activity of wild-type HisA (15). Nevertheless, we investigated whether ProFAR can still bind to HisAFcomIII and HisA-II by measuring its effect on the TrpF activity of both variants.…”

“…1 A) (15). The proximity of the side chain of aspartate 8, which is located at the C-terminal end of ␤-strand 1, to the ribose moiety of PRA (Fig.…”

“…Because the sequences of the N-terminal halves of HisA and HisAF are identical, it is very likely that aspartate 8 is also active as catalytic base in the TrpF reaction of HisAFcomIII. Also, the same aspartate 8 in HisA, as well as the corresponding cysteine 7 in tmTrpF, are the catalytic bases in the wild-type isomerisation reactions (15). In tmTrpF, aspartate 126 at the C-terminal end .…”

“…Along these lines, we earlier used random mutagenesis and selection in vivo to establish the catalytic activity of phosphoribosyl anthranilate (PRA) isomerase (TrpF) on the (␤␣) 8 -barrel scaffold of NЈ-[(5Ј-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA) from Thermotoga maritima (13,14). The wild-type TrpF and HisA enzymes catalyze mechanistically related isomerisation reactions of aminoaldoses into the corresponding aminoketoses within tryptophan and histidine biosynthesis (15). However, their substrates, Schiff base intermediates, and products contain a different substituent linked to the nitrogen atom of the corresponding aminosugars (Fig.…”

Mimicking enzyme evolution by generating new (βα) 8 -barrels from (βα) 4 -half-barrels

“…The pairs of N-terminal halves (designated HisF-N and HisA-N), which consist of the first four (␤␣) units, and the pairs of C-terminal halves (designated HisF-C and HisA-C), which consist of the last four (␤␣) units, display sequence identities between 16% and 26% and rms deviation values of their main-chain nonhydrogen atoms between 1.4 and 2.1 Å (10,14). Moreover, the catalytically essential aspartate residues of both HisF and HisA are located at equivalent positions at the C-terminal ends of the respective strands ␤1 (within HisF-N and HisA-N) and ␤5 (within HisF-C and HisA-C) (10,(15)(16)(17). When produced separately, the halfbarrels HisF-N and HisF-C are homodimeric proteins with native secondary and tertiary structures but without measurable catalytic activity.…”