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N-Succinylamino Acid Racemases

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N-Succinylamino Acid Racemases 1 N-succinylamino acid racemases: enzymatic properties and biotechnological 2 applications 3 4 Sergio Martínez-Rodríguez1,2,*, Pablo Soriano-Maldonado3, Jose Antonio Gavira2 5 6 1Departamento de Bioquímica y Biología Molecular III e Inmunología. Universidad de 7 Granada. Facultad de Medicina. Spain. 2Laboratorio de Estudios Cristalográficos. CSIC. 8 18100. Granada. Spain. 3Centro Nacional de Investigaciones Oncológicas. Madrid. 9 Spain. 10 11 *e-mail of correspondence: [email protected] 12 13 14 15 Keywords: N-succinylamino acid racemase; N-acetyl-amino acid racemase; N-acyl- 16 amino acid racemase; racemase; amino acid; enzymatic cascade 17 18 19 Abbreviations: N-succinylamino acid racemase (NSAR); o-succinylbenzoate synthase 20 (OSBS); N-succinylamino acid racemase/o-succinylbenzoate synthase subfamily 21 (NSAR/OSBS); N-acetyl-amino or N-acyl-amino acid racemase (NAAR); N- 22 substituted-amino acid (NxA); N-substituted-amino acid racemase (NxAR); Kinetic 23 resolution (KR); Dynamic kinetic resolution (DKR); N-acetyl- (NA); N-acetyl-amino 24 acid (NAA); N-succinyl- (NS); N-succinyl-amino acid (NSA); N-carbamoyl- (NC); N- 25 carbamoyl-amino acid (NCAA); N-chloroacetyl- (NCh); N-chloroacetyl-amino acid 26 (NChAA); N-formyl- (NF); N-formyl-amino acid (NFAA); N-butyril (NBt); N-butyril 27 amino acid (NBtA); N-propionyl (NPr); N-propionyl amino acid (NPrA); N-benzoyl 28 (NBzA); N-benzoyl amino acid (NBzA); (1R,6R)-2-succinyl-6-hydroxy-2,4- 29 cyclohexadiene-1-carboxylate (SHCHC); Amycolatopsis sp. TS-1-60 NSAR 30 (AmyNSAR); Geobacillus kaustophilus NSAR (GkaNSAR); Geobacillus 31 stearothermophilus CECT 49 NSAR (GstNSAR); Streptomyces atratus Y-53 NSAR 32 (SatNSAR); Sebekia benihana NSAR (SebeNSAR); Chloroflexus aurantiacus NSAR 33 (CauNSAR); Thermus thermophilus NSAR (TteNSAR); Deinococcus radiodurans 34 NSAR (DraNSAR); Exiguobacterium sp. AT1b OSBS (ExiOSBS); Alicyclobacillus 35 acidocaldarius OSBS (AacOSBS); Enterococcus faecalis V583 NSAR (EfaNSAR); 36 Listeria innocua Clip11262 NSAR (LinNSAR); Lysinibacillus varians GY32 NSAR 37 (LvaNSAR); Roseiflexus castenholzii HLO8 NSAR (RcaNSAR); Amycolatopsis 38 mediterranei S699 NSAR (AmedNSAR). 39 40 ABSTRACT 41 42 The N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) 43 subfamily from the enolase superfamily contains different enzymes showing 44 promiscuous N-substituted-amino acid racemase (NxAR) activity. These enzymes were 45 originally named as N-acylamino acid racemases because of their industrial application. 46 Nonetheless, they are pivotal in several enzymatic cascades due to their versatility to 47 catalyze a wide substrate spectrum, allowing the production of optically pure D- or L- 48 amino acids from cheap precursors. These compounds are of paramount economic 49 interest, since they are used as food additives, in the pharmaceutical and cosmetics 50 industries and/or as chiral synthons in organic synthesis. Despite its economic 51 importance, the discovery of new N-succinylamino acid racemases has become elusive, 52 since classical sequence-based annotation methods proved ineffective in their 53 identification, due to a high sequence similarity among the members of the enolase 54 superfamily. During the last decade, deeper investigations into different members of the 55 NSAR/OSBS subfamily have shed light on the classification and identification of 56 NSAR enzymes with NxAR activity of biotechnological potential. This review aims to 57 gather the dispersed information on NSAR/OSBS members showing NxAR activity 58 over recent decades, focusing on their biotechnological applications and providing 59 practical advice to identify new enzymes. 60 61 Introduction 62 63 As for other enzymatic racemization methodologies involved in dynamic kinetic 64 resolution (DKR) processes, the driving force boosting N-succinylamino acid racemase 65 (NSAR)1 research is most likely the existence of a previous enzymatic kinetic resolution 66 (KR) method for the production of optically pure amino acids. This KR process was 1 We will use different acronyms (NSAR, NAAR) through the manuscript to refer to enzymes belonging to the N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) subfamily. 67 already known since middle of the twentieth century [1], and takes advantage of the 68 stereospecificity of acylases (or aminoacylases, E.C. 3.5.1.14) to produce optically pure 69 amino acids from racemic mixtures of N-acetylamino acids (NAAs) [2, and references 70 therein]. Evonik (Degussa) industries already manufactured L-methionine at industrial 71 level in the 70`s using the “Acylase process” [3]. Furthermore, patents on the chemical 72 racemization of NAAs by different companies clearly illustrate the interest to develop a 73 chemo-enzymatic DKR before the eighties (e.g., Ajinomoto or Evonik/Degussa [4,5]). 74 Takeda Pharmaceutical Co Ltd discovered the N-acylamino acid racemase (NAAR) 75 activity in different microorganisms by the end of the 80’s, demonstrating a potential 76 NAAR/acylase enzymatic tandem, also providing a new DKR approach for optically 77 pure D- and L-amino acids [6-8]. The industrial relevance of these enzymes is clear by 78 the extensive number of patents issued [6-43] (Table 1). 79 80 To the best of our knowledge, the first paper dealing with an NSAR enzyme was not 81 published until the 90s. Tokuyama’s group described NAAR activity in some 82 actinomycetes strains belonging to the genus Streptomyces, Actinomadura, 83 Actinomyces, Jensenia, Amycolatopsis and Sebekia. However, they did not find this 84 activity in other bacteria, yeasts or fungi, from more than 44000 strains tested [44,45]. 85 From the organisms described in that work, enzymes belonging to Streptomyces atratus 86 Y-53 (SatNSAR) [46], Sebekia benihana (SebeNSAR) [22-24] and Amycolatopsis [47- 87 49] were characterized and used (Table 1). Whereas the principal interest of these 88 enzymes was the racemization of NAAs, their ability to racemize different N- 89 substituted-amino acids (NxAs) was also soon reported, showing them to be active 90 towards N-acetyl (NAAs), N-carbamoyl (NCAs), N-chloroacetyl (NChAs), N-butyril 91 (NBtAs), N-propionil (NPrAs), N-benzoyl (NBzAs) or N-formyl-amino acids (NFAs), 92 among others [46,47]. Despite the potential DKR application of these enzymes, only a 93 discrete number of NAAR enzymes were identified and/or characterized in the 94 following years, mainly from Amycolatopsis and Deinococcus genus, mostly boosted by 95 their industrial interest [25, 50-53, see also Table 1] or for structural characterization 96 [54,55]. Additional actinomycetes strains were shown to possess NAAR activity, 97 although no further information has been reported on the enzymes from these 98 microorganisms [56]. NSAR was already shown to be a member of the functional 99 diverse enolase superfamily before the beginning of the new millennium [51]. Several 100 works at the beginning of the 21st century focused on deciphering the natural function of 101 NSARs and some evolutionary aspects [51,57,58, 69-76] (see below), allowing to 102 localize new NSARs in Bacillus/Geobacillus genera. Some of the NSARs belonging to 103 this genera have been highly characterized and/or applied to the production of optically 104 pure amino acids [27-38,59,61-68]. A different N-succinyl arginine/lysine racemase 105 from Bacillus cereus ATCC 14579, highly specific for NS-Arg and NS-Lys has been 106 reported, although it does not belong to the NSAR group reviewed in this paper [59]. 107 The characterization of an NSAR from Chloroflexus aurantiacus (CauNSAR) [26] and 108 the determination of the crystal structure of Thermus thermophilus NSAR (TteNSAR) 109 [60], further expand the organisms in which NSAR activity has been found. Finally, 110 Exiguobacterium, Lysinibacillus, Roseiflexus, Enterococcus or Listeria genera have also 111 been reported to present NSAR activity, although they have been studied in less detail 112 from the biotechnological point of view [72-74] 113 114 Natural function and evolutionary aspects on NSAR enzymes 115 116 After one decade using the industrially-biased NAAR nomenclature, Gerlt’s group gave 117 an important step towards understanding the natural function of NSAR, suggesting that 118 the “correct” function of Amycolatopsis sp. TS-1-60 NAAR (AmyNSAR) was the o- 119 succinylbenzoate synthase (OSBS) reaction in menaquinone biosynthesis [51], due to 120 the sequence similarity to Bacillus subtilis OSBS. The NAAR activity was suggested as 121 a secondary and promiscuous activity of OSBS, with no impact on the organism in its 122 metabolic niche (Figure 1). Despite the significance of this work in the comprehension 123 of the promiscuous NSAR activity, the NAAR nomenclature continued to be used for 124 several years in the literature. Another milestone in the understanding of the function of 125 NSAR enzymes was achieved in 2006 [57]. Geobacillus kaustophilus was shown to 126 contain a member of the enolase superfamily with NSAR activity (GkaNSAR), 127 homolog to the well-characterized enzymes from Amycolatopsis. GkaNSAR was 128 encoded in a new gene cluster, different to the menaquinone biosynthesis operon in 129 which OSBS is included [51] (Figure 2). A similar cluster was identified for 130 Amycolatopsis and Thermus genera [57] (Figure 2). After two decades of the discovery 131 of NSARs, the authors concluded that GkaNSAR constitutes part of a novel, irreversible 132 pathway for the conversion of D- to L-amino acids (Figure 2, lower panel), different to 133 the
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