The crystal structure of isopenicillin N synthase with a dipeptide substrate analogue

Homocysteine in Protein Structure/Function and Human Disease

“…This shows that Hcy incorporation into protein in place of Met does not lead to breakage of peptide bonds and that Hcy is compatible with protein structure. While Hcy-oxytocin is devoid of biological activity of normal oxytocin [247], AhCV is a substrate for isopenicillin synthase, which oxidizes AhCV to a δ-lactam, a higher homolog of the γ-lactam arising from the oxidation of the natural cysteine-containing precursor [248]. Resulting Hcycontaining peptides are chemically stable.…”

The Interaction of Isopenicillin N Synthase with Homologated Substrate Analogues δ‐(L‐α‐Aminoadipoyl)‐L‐homocysteinyl‐D‐Xaa Characterised by Protein Crystallography

“…Global deprotection with TFA29 gave AhCV ( 4 ) or AhCmC ( 12 ) as their TFA salts, from which the pure tripeptides were obtained by reversed‐phase HPLC. Deprotection of the S ‐methylcysteine derivative 21 also gave rise to a significant quantity of the cyclic thioester 22 ,21 which is presumably formed through thiol‐mediated cleavage of the amide bond under the acidic conditions of the deprotection reaction.…”

“… Synthesis of tripeptide analogues 4 and 12 ; a) NaOH, (Boc) 2 O, H 2 O/ t BuOH, RT, 92 %; b) PMBCl, Et 3 N, DMF, 38 °C, 60 %; c) Na, NH 3(l) , −78 °C; d) PMBCl, −78 °C to RT, 83 % (over two steps); e) isobutyl chloroformate, Et 3 N, THF/H 2 O, 0 °C, 90 %; f) 18 or 19 , EDCI, HOBt, Et 3 N, CH 2 Cl 2 , RT, 66 % ( 20 )/46 % ( 21 ); g) TFA, anisole, reflux, RP‐HPLC 46 % ( 4 )/12 % ( 12 ); L ‐AA=δ‐( L ‐α‐aminoadipoyl). Insert: the cyclic thioether ( S )‐2‐amino‐6‐oxo‐6‐[(( S )‐2‐oxotetrahydrothiophen‐3‐yl)amino]hexanoic acid ( 22 ) was isolated as the major product from TFA‐mediated deprotection of 21 21…”

“…15 Crystal structures of IPNS with its natural substrate and analogues have elucidated different modes of substrate and analogue binding, and revealed the influence of active site geometry on reactivity 9. 16–21 The application of hyperbaric oxygenation to trigger reaction within crystalline IPNS has rendered further detail, allowing the enzyme mechanism to be studied step‐by‐step within the crystalline protein 10. 22–26…”

The crystal structure of an isopenicillin N synthase complex with an ethereal substrate analogue reveals water in the oxygen binding site

“…AC‐ d ‐thia‐ allo ‐isoleucine (ACt a I, 9 ) [20] and AC‐ d ‐methionine [49]), in which the affinity of sulfur for iron means that the sulfide S coordinates to the metal; (ii) with smaller substrate analogues (e.g. AC‐Gly and AC‐ d ‐Ala,[28] AC‐ d ‐α‐aminobutyrate [26], AC‐ d ‐vinylglycine [23] and the dipeptide δ‐ l ‐α‐aminoadipoyl‐ l ‐homocysteine (AhC) [50]), where the smaller side‐chain leaves room for a second water ligand to bind to iron opposite Asp216; and (iii) with lll ‐configured substrates (e.g. AC‐ l ‐hexafluorovaline [51] and AC‐ l ‐2‐amino‐3,3‐dideuteriobutyrate [52]), where the different substrate stereochemistry permits an additional water ligand at iron.…”